Compositions and methods for enhanced mucosal delivery of Y2 receptor-binding peptides and methods for treating and preventing obesity

ABSTRACT

Pharmaceutical compositions and methods are described comprising at least one Y2 receptor-binding peptide, such as peptide YY(PYY), Neuropeptide Y (NPY) or Pancreatic Peptide (PP) and one or more mucosal delivery-enhancing agents for enhanced nasal mucosal delivery of the peptide YY, for treating a variety of diseases and conditions in mammalian subjects, including obesity.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This is a continuation application and claims priority under 35U.S.C. §120 of co-pending U.S. patent application Ser. No. 10/745,069filed Dec. 23, 2003, which is a continuation-in-part of U.S. patentapplication Ser. No. 10/322,266, filed Dec. 17, 2002, and claimspriority under 35 U.S.C. §119 (e) of U.S. Provisional Application No.60/493,226, filed Aug. 7, 2003, U.S. Provisional Application No.60/501,170, filed Sep. 8, 2003, U.S. Provisional Application No.60/510,785, filed Oct. 10, 2003, U.S. Provisional Application No.60/517,290, filed Nov. 4, 2003; U.S. Provisional Application No.60/518,812, filed on Nov. 10, 2003; and PCT/US03/40538, filed on Dec.17, 2003; the entire contents of these applications are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

[0002] The teachings of all the references cited in the presentspecification are incorporated in their entirety by reference.

[0003] Obesity and its associated disorders are common and very seriouspublic health problems in the United States and throughout the world.Upper body obesity is the strongest risk factor known for type-2diabetes mellitus, and is a strong risk factor for cardiovasculardisease. Obesity is a recognized risk factor for hypertension,arteriosclerosis, congestive heart failure, stroke, gallbladder disease,osteoarthritis, sleep apnea, reproductive disorders such as polycysticovarian syndrome, cancers of the breast, prostate, and colon, andincreased incidence of complications of general anesthesia. It reduceslife-span and carries a serious risk of co-morbidities above, as welldisorders such as infections, varicose veins, acanthosis nigricans,eczema, exercise intolerance, insulin resistance, hypertensionhypercholesterolemia, cholelithiasis, orthopedic injury, andthromboembolic disease. Obesity is also a risk factor for the group ofconditions called insulin resistance syndrome, or “Syndrome X.”

[0004] It has been shown that certain peptides that bind to the Y2receptor when administered peripherally to a mammal induce weight loss.The Y2 receptor-binding peptides are neuropeptides that bind to the Y2receptor. Neuropeptides are small peptides originating from largeprecursor proteins synthesized by peptidergic neurons andendocrine/paracrine cells. Often the precursors contain multiplebiologically active peptides. There is great diversity of neuropeptidesin the brain caused by alternative splicing of primary gene transcriptsand differential precursor processing. The neuropeptide receptors serveto discriminate between ligands and to activate the appropriate signals.These Y2 receptor-binding peptides belong to a family of peptidesincluding peptide YY (PYY), neuropeptide Y (NPY) and pancreatic peptide(PP).

[0005] NPY is a 36-amino acid peptide and is the most abundantneuropeptide to be identified in mammalian brain. NPY is an importantregulator in both the central and peripheral nervous systems andinfluences a diverse range of physiological parameters, includingeffects on psychomotor activity, food intake, central endocrinesecretion, and vasoactivity in the cardiovascular system. Highconcentrations of NPY are found in the sympathetic nerves supplying thecoronary, cerebral, and renal vasculature and have contributed tovasoconstriction. NPY binding sites have been identified in a variety oftissues, including spleen, intestinal membranes, brain, aortic smoothmuscle, kidney, testis, and placenta.

[0006] Neuropeptide Y (NPY) receptor pharmacology is currently definedby structure activity relationships within the pancreatic polypeptidefamily. This family includes NPY, which is synthesized primarily inneurons; PYY, which is synthesized primarily by endocrine cells in thegut; and PP, which is synthesized primarily by endocrine cells in thepancreas. These approximately 36 amino acid peptides have a compacthelical structure involving a “PP-fold” in the middle of the peptide.Specific features include a polyproline helix in residues 1 through 8, aβ-turn in residues 9 through 14, an α-helix in residues 15 through 30,an outward-projecting C-terminus in residues 30 through 36, and acarboxyl terminal amide, which appears to be critical for biologicalactivity. The peptides have been used to define at least five receptorsubtypes known as Y1, Y2, Y3, Y4 and Y5. Y1 receptor recognition by NPYinvolves both N- and C-terminal regions of the peptide; exchange ofGln³⁴ with Pro³⁴ is fairly well tolerated. Y2 receptor recognition byNPY depends primarily upon the four C-terminal residues of the peptide(Arg³³—Gln³⁴—Arg³⁵—Tyr³⁶—NH2) preceded by an amphipathic an OL-helix;exchange of Gln³⁴ with Pro³⁴ is not well tolerated. One of the keypharmacological features which distinguish Y1 and Y2 is the fact thatthe Y2 receptor (and not the Y 1 receptor) has high affinity for the NPYpeptide carboxyl-terminal fragment NPY-(13-36) and the PYY fragmentPYY(22-36).

[0007] It has been shown that a 36 amino acid peptide called PeptideYY(1-36)[PYY(1-36)] [YPIKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY, SEQ ID NO.:1]. when administered peripherally by injection to an individualproduces weight loss and thus can be used as a drug to treat obesity andrelated diseases, Morley, J. Neuropsychobiology 21:22-30 (1989). It waslater found that to produce this effect PYY bound to a Y2 receptor, andthe binding of a Y2 agonist to the Y2 receptor caused a decrease in theingestion of carbohydrate, protein and meal size, Leibowitz, S. F. etal. Peptides, 12:1251-1260 (1991). An alternate molecular form of PYY isPYY(3-36) IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY [SEQ ID NO.: 2], Eberlein,Eysselein et al. Peptides 10: 797-803, 1989). This fragment constitutesapproximately 40% of total PYY-like immunoreactivity in human and canineintestinal extracts and about 36% of total plasma PYY immunoreactivityin a fasting state to slightly over 50% following a meal. It isapparently a dipeptidyl peptidase-IV (DPP4) cleavage product of PYY.PYY3-36 is reportedly a selective ligand at the Y2 and Y5 receptors,which appear pharmacologically unique in preferring N-terminallytruncated (i.e. C-terminal fragments of) NPY analogs. It has also beenshown that a PYY fragment having only residues 22-36 will still bind tothe Y2 receptor. However, if any of the carboxyl terminus of the peptideis cleaved, the peptide looses its ability to bind to the Y2 receptor.Hence a PYY agonist is a peptide, which has a partial sequence offull-length PYY and is able to bind to a Y2 receptor in the arcuatenucleus of the hypothalamus. Hereinafter the term PYY refers tofull-length PYY and any fragment of PYY that binds to a Y2 receptor.

[0008] It is known that PYY and PYY3-36 can be administered byintravenous infusion or injection to treat life-threatening hypotensionas encountered in shock, especially that caused by endotoxins (U.S. Pat.No. 4,839,343), to inhibit proliferation of pancreatic tumors in mammalsby perfusion, parenteral, intravenous, or subcutaneous administration,and by implantation (U.S. Pat. No. 5,574,010) and to treat obesity(Morley, J. Neuropsychobiology 21:22-30 (1989) and U.S. patentapplication 20020141985). It is also claimed that PYY can beadministered by parenteral, oral, nasal, rectal and topical routes todomesticated animals or humans in an amount effective to increase weightgain of said subject by enhancing gastrointestinal absorption of asodium-dependent cotransported nutrient (U.S. Pat. No. 5,912,227).However, for the treatment of obesity and related diseases, includingdiabetes, the mode of administration has been limited to intravenous IVinfusion with no effective formulations optimized for alternativeadministration of PYY3-36. None of these prior art teachings provideformulations that contain PYY or PYY(3-36) combined with excipientsdesigned to enhance mucosal (i.e., nasal, buccal, oral) delivery nor dothey teach the value of endotoxin-free Y2-receptor binding peptideformulations for non-infused administration. Thus, there is a need todevelop formulations and methods for administering PYY3-36.

SUMMARY OF THE INVENTION

[0009] The present invention fulfills the foregoing needs and satisfiesadditional objects and advantages by providing novel, effective methodsand compositions for mucosal, especially intranasal, delivery of a Y2receptor-binding peptide such as PYY, Pancreatic Peptide (PP) and NPY,to treat obesity, induce satiety in an individual and to promoteweight-loss in an individual and prevent or cure diabetes. In certainaspects of the invention, the Y2 receptor-binding peptide is deliveredin formulations to the intranasal mucosa so as to be able to increasethe concentration of the Y2 receptor-binding peptide by at least 5 pmol,preferably by at least 10 pmol, in the blood plasma of a mammal when adose of the formulations of the Y2 receptor agonist is administeredintranasally. Furthermore preferred formulations would be able to raisethe concentration of the Y2 receptor-binding peptide in the plasma of amammal by 10 pmol, preferably 20 pmol, when the Y2 receptor-bindingpeptide is administered intranasally. When 150 μg is administeredintranasally the preferred formulation would be able to raise theconcentration of the Y2 receptor agonist in the plasma of the mammal byat least 40 pmol per liter of plasma. When 200 μg of the Y2receptor-binding peptide is administered intranasally, the formulationsof the present invention induce at least 80 pmol, per liter of plasmaincrease of the Y2 receptor-binding peptide. In preferred embodiments,the elevated concentrations of the Y2-receptor-binding peptide remainselevated in the plasma of the mammal for at least 30 minutes, preferablyat least 60 minutes following a single intranasal dose of the Y2receptor-binding peptide.

[0010] Preferably the Y2 receptor-binding peptide is a PP, PYY or NPYpeptide and the mammal is a human. In a most preferred embodiment the Y2receptor-binding peptide is a PYY peptide, preferably PYY(3-36) and themammal is human.

[0011] The present invention is also related to a Y2 receptor-bindingpeptide formulation that is able to raise the concentration of the Y2receptor-binding peptide in the blood plasma of a mammal by at least 5pM when a dose containing at least 50 μg of the Y2 receptor-bindingpeptide is administered to the mammal. In preferred embodiments, theelevated concentrations of the Y2-receptor-binding peptide remainselevated in the plasma of the mammal for at least 30 minutes, preferablyat least 60 minutes following a single intranasal dose of the Y2receptor-binding peptide.

[0012] The present invention is also related to a Y2 receptor-bindingpeptide formulation that is able to raise the concentration of the Y2receptor-binding peptide in the blood-plasma of a mammal by at least 20pM when a dose containing at least 100 μg of the Y2 receptor-bindingpeptide is administered to the mammal. In preferred embodiments, theelevated concentrations of the Y2-receptor-binding peptide remainselevated in the plasma of the mammal for at least 30 minutes, preferablyat least 60 minutes following a single intranasal dose of the Y2receptor-binding peptide.

[0013] The present invention is also related to a Y2 receptor-bindingpeptide formulation that when administered intranasally to a mammal isable to raise the concentration of the Y2 receptor-binding peptide inblood plasma of the mammal by at least 30 pM when a dose containing atleast 150 μg of the Y2 receptor-binding peptide is administered. Inpreferred embodiments, the elevated concentrations of theY2-receptor-binding peptide remains elevated in the plasma of the mammalfor at least 30 minutes, preferably at least 60 minutes following asingle intranasal dose of the Y2 receptor-binding peptide. Preferablythe mammal is a human.

[0014] The present invention is also related to a Y2 receptor-bindingpeptide formulation that when administered intranasally to a mammal isable to raise the concentration of the Y2 receptor-binding peptide by atleast 60 pM when a dose containing at least 200 μg is administered tothe mammal. In preferred embodiments, the elevated concentrations of theY2-receptor-binding peptide remains elevated in the plasma of the mammalfor at least 30 minutes, preferably at least 60 minutes following asingle intranasal dose of the Y2 receptor-binding peptide. Preferablythe mammal is a human.

[0015] The present invention is also directed to an intranasalformulation of a Y2 receptor-agonist that is substantially free ofproteins or polypeptides that stabilize the formulation. In particular,the preferred formulation is free of such proteins as albumin, andcollagen-derived proteins such as gelatin.

[0016] In other aspects of the present invention a transmucosal Y2receptor-binding peptide formulation is comprised of a Y2receptor-binding peptide, water and a solubilizing agent having a pH of3-6.5. In a preferred embodiment, the solubilization agent is acyclodextrin.

[0017] In another embodiment of the present invention a transmucosal Y2receptor-binding peptide formulation is comprised of a Y2receptor-binding peptide, water, a solubilizing agent, preferably acyclodextrin, and at least one polyol, preferably 2 polyols. Inalternate embodiments the formulation may contain one or all of thefollowing: a chelating agent, a surface-acting agent and a bufferingagent.

[0018] In another embodiment of the present invention the formulation iscomprised of a Y2 receptor-binding peptide, water, chelating agent and asolubilization agent.

[0019] In another embodiment of the present invention the formulation iscomprised of a Y2 receptor-binding peptide, water and a chelating agenthaving a pH of 3-6.5.

[0020] In another embodiment of the present invention the formulation iscomprised of a Y2 receptor-binding peptide, water, chelating agent andat least one polyol, preferably two polyols. Additional embodiments mayinclude one or more of the following: a surface-active agent, asolubilizing agent and a buffering agent.

[0021] In another embodiment of the present invention the formulation iscomprised of a Y2 receptor-binding peptide, water, and at least twopolyols, such as lactose and sorbitol. Additional agents, which can beadded to the formulation, include, but are not limited to, asolubilization agent, a chelating agent, one or more buffering agentsand a surface-acting agent.

[0022] The enhancement of intranasal delivery of a Y2 receptor-bindingpeptide agonist according to the methods and compositions of theinvention allows for the effective pharmaceutical use of these agents totreat a variety of diseases and conditions in mammalian subjects.

[0023] The present invention fills this need by providing for a liquidor dehydrated Y2 receptor-binding peptide formulation wherein theformulation is substantially free of a stabilizer that is a polypeptideor a protein. The liquid PYY formulation is comprised of water, PYY andat least one of the following additives selected from the groupconsisting of polyols, surface-active agents, solubilizing agents andchelating agents. The pH of the formulation is preferably 3 to about7.0, referably 4.5 to about 6.0, most preferably about 5.0±0.03.

[0024] Another embodiment of the present invention is an aqueous Y2receptor-binding formulation of the present invention is comprised ofwater, a Y2 receptor-binding peptide, a polyol and a surface-activeagent wherein the formulation has a pH of about 3 to about 6.5, and theformulation is substantially free of a stabilizer that is a protein orpolypeptide.

[0025] Another embodiment of the present invention is an aqueous Y2receptor-binding peptide formulation comprised of water, Y2receptor-binding peptide, a polyol and a solubilizing agent wherein theformulation has a pH of about 3.0 to about 6.5, and the formulation issubstantially free of a stabilizer that is a protein or polypeptide.

[0026] Another embodiment of the present invention is an aqueous Y2receptor-binding peptide formulation comprised of water, Y2receptor-binding peptide, a solubilizing agent and a surface-activeagent wherein the formulation has a pH of about 3.0 to about 6.5, andthe formulation is substantially free of a stabilizer that is a proteinor polypeptide.

[0027] Another embodiment of the invention is a aqueous Y2receptor-binding peptide formulation comprised of water, a Y2receptor-binding peptide, a solubilizing agent, a polyol and asurface-active agent wherein the formulation has a pH of about 3.0 toabout 6.5, and the formulation is substantially free of a stabilizerthat is a protein or polypeptide.

[0028] In another aspect of the present invention, the stable aqueousformulation is dehydrated to produce a dehydrated Y2 receptor-bindingpeptide formulation comprised of Y2 receptor-binding peptide and atleast one of the following additives selected from the group consistingof polyols, surface-active agents, solubilizing agents and chelatingagents, wherein said dehydrated Y2 receptor-binding peptide formulationis substantially free of a stabilizer that is a protein or polypeptidesuch as albumin, collagen or collagen-derived protein such as gelatin.The dehydration can be achieved by various means such as lyophilization,spray-drying, salt-induced precipitation and drying, vacuum drying,rotary evaporation, or supercritical CO₂ precipitation.

[0029] In one embodiment, the dehydrated Y2 receptor-binding peptide iscomprised of Y2 receptor-binding peptide, a polyol and a solubilizingagent, wherein the formulation is substantially free of a stabilizerthat is a protein.

[0030] In another embodiment, the dehydrated Y2 receptor-binding peptideformulation is comprised of a Y2 receptor-binding peptide, a polyol, anda surface-active agent wherein the Y2 receptor-binding peptideformulation is substantially free of a stabilizer that is a protein orpolypeptide.

[0031] In another embodiment, the dehydrated Y2 receptor-binding peptideformulation is comprised of a Y2 receptor-binding peptide, asurface-active agent, and a solubilizing agent wherein the Y2receptor-binding peptide formulation is substantially free of astabilizer that is a protein or polypeptide.

[0032] In another embodiment of the present invention, the dehydrated Y2receptor-binding peptide formulation is comprised of a Y2receptor-binding peptide, a polyol, a surface-active agent and asolubilizing agent wherein the Y2 receptor-binding peptide formulationis substantially free of a stabilizer that is a protein or polypeptide.

[0033] Any solubilizing agent can be used but a preferred one isselected from the group consisting of hydroxypropyl-β-cyclodextran,sulfobutylether-β-cyclodextran, methyl-β-cyclodextrin and chitosan.

[0034] Generally a polyol is selected from the group consisting oflactose, sorbitol, trehalose, sucrose, mannose and maltose andderivatives and homologs thereof.

[0035] A satisfactory surface-active agent is selected from the groupconsisting of L-α-phospharidycholine didecanoyl (DDPC), polysorbate 20(Tween 20), polysorbate 80 (Tween 80), polyethylene glycol (PEG), cetylalcohol, polyvinylpyrolidone (PVP), polyvinyl alcohol (PVA), lanolinalcohol, and sorbitan monooleate.

[0036] In a preferred formulation, the Y2 receptor-binding peptideformulation is also comprised of a chelating agent such as ethylenediamine tetraacetic acid (EDTA) or ethylene glycol tetraacetic acid(EGTA). Also a preservative such as chlorobutanol or benzylkoniumchloride can be added to the formulation to inhibit microbial growth.

[0037] The pH is generally regulated using a buffer such as sodiumcitrate and citric acid, and sodium acetate and acetic acid. Analternative buffer would be acetic acid and sodium acetate or succinicacid and sodium hydroxide.

[0038] The preferred Y2 receptor-binding peptide is a PYY, PP or NPYpeptide, preferably a PYY(3-36) peptide.

[0039] The present invention also comprehends a formulation wherein theconcentration of the Y2 receptor-binding peptide is 0.1-15.0 mg/mL,preferably 1.0-2 mg/mL and the pH of the aqueous solution is 3.0-6.5preferably about 5.0±0.3.

[0040] The present invention further includes Y2 receptor-bindingpeptide formulation wherein the concentration of the polyol is betweenabout 0.1% and 10% (w/v) and additionally wherein the concentration ofthe polyol is in the range from about 0.1% to about 3% (w/v).

[0041] The instant invention also includes a formulation, wherein theconcentration of the surface-active agent is between about 0.00001% andabout 5%(w/v), and wherein the concentration of the surface-active agentis between about 0.0002% and about 0.1% (w/v).

[0042] The instant invention also includes a formulation, wherein theconcentration of the solubilzation agent is 1%-10% (w/v), and whereinthe concentration of the solubilizing agent is 2% to 5% (w/v).

[0043] The finished solution can be filtered and freeze-dried,lyophilized, using methods well known to one of ordinary skill in theart, and by following the instructions of the manufacturer of thelyophilizing equipment. This produces a dehydrated Y2 receptor-bindingpeptide formulation substantially free of a stabilizer that is aprotein.

[0044] In another embodiment of the present invention, a Y2receptor-binding peptide formulation is comprised of an Y2receptor-binding peptide and a pharmaceutically acceptable carrierwherein the Y2 receptor-bind peptide formulation has at least 1%,preferably 3% and most preferably at least 6% higher permeation in an invitro tissue permeation assay than a control formulation consisting ofwater, sodium chloride, a buffer and the Y2 receptor-binding peptide, asdetermined by the transepithelial electrical resistance assay shown inExamples 2 & 7. In a preferred embodiment, the Y2 receptor-bindingformulation is further comprised of at least one excipient selected fromthe group consisting of a surface-active agent, a solubilization agent,a polyol, and a chelating agent. Preferably the Y2 receptor-bindingpeptide is a PYY peptide, an NPY peptide or a PP peptide.

[0045] In another embodiment of the present invention a Y2receptor-binding petide formulation is provided that is capable ofraising the concentration of the Y2 receptor-binding peptide in theplasma of a mammal by at least 5 preferably 10, 20 40, 60, 80 or morepmoles per liter of plasma when 100 μL of the formulation isadministered intranasally to said mammal.

[0046] In exemplary embodiments, the enhanced delivery methods andcompositions of the present invention provide for therapeuticallyeffective mucosal delivery of the Y2 receptor-binding peptide agonistfor prevention or treatment of obesity and eating disorders in mammaliansubjects. In one aspect of the invention, pharmaceutical formulationssuitable for intranasal administration are provided that comprise atherapeutically effective amount of a Y2 receptor-binding peptide andone or more intranasal delivery-enhancing agents as described herein,which formulations are effective in a nasal mucosal delivery method ofthe invention to prevent the onset or progression of obesity or eatingdisorders in a mammalian subject. Nasal mucosal delivery of atherapeutically effective amount of a Y2 receptor-binding peptideagonist and one or more intranasal delivery-enhancing agents yieldselevated therapeutic levels of the Y2 receptor-binding peptide agonistin the subject and inhibits food intake in the mammalian subject,reducing symptoms of obesity or an eating disorder.

[0047] The enhanced delivery methods and compositions of the presentinvention provide for therapeutically effective mucosal delivery of a Y2receptor-binding peptide for prevention or treatment of a variety ofdiseases and conditions in mammalian subjects. Y2 receptor-bindingpeptide can be administered via a variety of mucosal routes, for exampleby contacting the Y2 receptor-binding peptide to a nasal mucosalepithelium, a bronchial or pulmonary mucosal epithelium, the oral buccalsurface or the oral and small intestinal mucosal surface. In exemplaryembodiments, the methods and compositions are directed to or formulatedfor intranasal delivery (e.g., nasal mucosal delivery or intranasalmucosal delivery).

[0048] In one aspect of the invention, pharmaceutical formulationssuitable for intranasal administration are provided that comprise atherapeutically effective amount of a Y2 receptor-binding peptideagonist and one or more intranasal delivery-enhancing agents asdescribed herein, which formulations are effective in a nasal mucosaldelivery method of the invention to prevent the onset or progression ofobesity, diabetes, cancer, or malnutrition or wasting related to cancerin a mammalian subject, or to alleviate one or more clinicallywell-recognized symptoms of obesity, as well as treating Alzheimer'sdisease, colon carcinoma, colon adenocarcinoma, pancreatic carcinoma,pancreatic adenocarcinoma, breast carcinoma.

[0049] In another aspect of the invention, pharmaceutical formulationsand methods are directed to administration of a Y2 receptor-bindingpeptide agonist in combination with vitamin E succinate. A Y2receptor-binding peptide agonist in combination with vitamin E succinatemay be administered to alleviate symptoms or prevent the onset or lowerthe incidence or severity of cancer, for example, colon adenocarcinoma,pancreatic adenocarcinoma, or breast cancer.

[0050] In another aspect of this invention, it was surprisingly foundthat the use of endotoxin-free Y2 receptor binding peptides, for examplePYY(3-36), produced increased mucosal delivery compared to peptides inwhich endotoxin is not removed. The use of endotxin-free Y2 receptorpeptides in pharmaceutical formulations is thus enabled foradministration by non-infusion routes, including mucosal delivery,nasal, oral, pulmonary, vaginal, rectal and the like.

[0051] The foregoing mucosal Y2 receptor-binding peptide formulationsand preparative and delivery methods of the invention provide improvedmucosal delivery of a Y2 receptor-binding peptide to mammalian subjects.These compositions and methods can involve combinatorial formulation orcoordinate administration of one or more Y2 receptor-binding peptideswith one or more mucosal delivery-enhancing agents. Among the mucosaldelivery-enhancing agents to be selected from to achieve theseformulations and methods are (A) solubilization agents; (B) chargemodifying agents; (C) pH control agents; (D) degradative enzymeinhibitors; (E) mucolytic or mucus clearing agents; (F) ciliostaticagents; (G) membrane penetration-enhancing agents (e.g., (i) asurfactant, (ii) a bile salt, (iii) a phospholipid or fatty acidadditive, mixed micelle, liposome, or carrier, (iv) an alcohol, (v) anenamine, (iv) an NO donor compound, (vii) a long-chain amphipathicmolecule (viii) a small hydrophobic penetration enhancer; (ix) sodium ora salicylic acid derivative; (x) a glycerol ester of acetoacetic acid(xi) a cyclodextrin or beta-cyclodextrin derivative, (xii) amedium-chain fatty acid, (xiii) a chelating agent, (xiv) an amino acidor salt thereof, (xv) an N-acetylamino acid or salt thereof, (xvi) anenzyme degradative to a selected membrane component, (xvii) an inhibitorof fatty acid synthesis, (xviii) an inhibitor of cholesterol synthesis;or (xiv) any combination of the membrane penetration enhancing agents of(i)-(xviii)); (H) modulatory agents of epithelial junction physiology,such as nitric oxide (NO) stimulators, chitosan, and chitosanderivatives; (I) vasodilator agents; (J) selective transport-enhancingagents; and (K) stabilizing delivery vehicles, carriers, supports orcomplex-forming species with which the Y2 receptor-binding peptide (s)is/are effectively combined, associated, contained, encapsulated orbound to stabilize the active agent for enhanced mucosal delivery.

[0052] In various embodiments of the invention, a Y2 receptor-bindingpeptide is combined with one, two, three, four or more of the mucosaldelivery-enhancing agents recited in (A)-(K), above. These mucosaldelivery-enhancing agents may be admixed, alone or together, with the Y2receptor-binding peptide, or otherwise combined therewith in apharmaceutically acceptable formulation or delivery vehicle. Formulationof a Y2 receptor-binding peptide with one or more of the mucosaldelivery-enhancing agents according to the teachings herein (optionallyincluding any combination of two or more mucosal delivery-enhancingagents selected from (A)-(K) above) provides for increasedbioavailability of the y2 receptor-binding peptide following deliverythereof to a mucosal surface of a mammalian subject.

[0053] Thus, the present invention is a method for suppressing apetite,promoting weight loss, decreasing food intake, or treating obesityand/or diabetes in a mammal comprising transmucosally administering aformulation comprised of a Y2 receptor-binding peptide, such that whenat 50 μg of the Y2 receptor is administered transmucosally to the mammalthe concentration of the Y2 receptor-binding peptide in the plasma ofthe mammal increases by at least 5 pmol, preferably at least 10 pmol perliter of plasma. Examples of such formulations are described above.

[0054] The present invention further provides for the use of a Y2receptor-binding peptide for the production of medicament for thetransmucosal, administration of a Y2 receptor-binding peptide forsuppressing apetite, promoting weight loss, decreasing food intake, ortreating obesity in a mammal such that when about 50 μg of the Y2receptor is administered transmucosally to the mammal the concentrationof the Y2 receptor-binding peptide in the plasma of the mammal increasesby at least 5 pmol per liter of plasma. When 100 μg of the Y2receptor-binding peptide is administered intranasally to the mammal, theconcentration of the Y2 receptor agonist increases by at least 20 pmolper liter of plasma in the mammal. When 150 μg is administeredintranasally, the concentration of the Y2 receptor-binding peptide inblood plasma of the mammal increases by at least 30 pM. When 200 μg isadministered intranasally, the concentration of the Y2 receptor-bindingpeptide in blood plasma of the mammal increases by at least 60 pM. Inpreferred embodiments, the elevated concentrations of theY2-receptor-binding peptide remains elevated in the plasma of the mammalfor at least 30 minutes, preferably at least 60 minutes following asingle intranasal dose of the Y2 receptor-binding peptide. Preferablythe mammal is a human.

[0055] A mucosally effective dose of peptide YY within thepharmaceutical formulations of the present invention comprises, forexample, between about 0.001 pmol to about 100 pmol per kg body weight,between about 0.01 pmol to about 10 pmol per kg body weight, or betweenabout 0.1 pmol to about 5 pmol per kg body weight. In further exemplaryembodiments, dosage of peptide YY is between about 0.5 pmol to about 1.0pmol per kg body weight. In a preferred embodiment an intranasal dosewill range from 50 μg to 400 μg, preferably 100 μg to 200 μg, mostpreferably about 100 μg to 150 μg. The pharmaceutical formulations ofthe present invention may be administered one or more times per day (forexample, before a meal), or 3 times per week or once per week forbetween one week and at least 96 weeks or even for the life of theindividual patient or subject. In certain embodiments, thepharmaceutical formulations of the invention are administered one ormore times daily, two times daily, four times daily, six times daily, oreight times daily.

[0056] Intranasal delivery-enhancing agents are employed which enhancedelivery of peptide YY into or across a nasal mucosal surface. Forpassively absorbed drugs, the relative contribution of paracellular andtranscellular pathways to drug transport depends upon the pKa, partitioncoefficient, molecular radius and charge of the drug, the pH of theluminal environment in which the drug is delivered, and the area of theabsorbing surface. The intranasal delivery-enhancing agent of thepresent invention may be a pH control agent. The pH of thepharmaceutical formulation of the present invention is a factoraffecting absorption of peptide YY via paracellular and transcellularpathways to drug transport. In one embodiment, the pharmaceuticalformulation of the present invention is pH adjusted to between about pH3.0 to 6.5. In a further embodiment, the pharmaceutical formulation ofthe present invention is pH adjusted to between about pH 3.0 to 5.0. Ina further embodiment, the pharmaceutical formulation of the presentinvention is pH adjusted to between about pH 4.0 to 5.0. Generally, thepH is 5.0±0.3.

BRIEF DESCRIPTION OF THE DRAWINGS

[0057]FIG. 1 shows the stability of PYY3-36 at high temperature (40° C.)at various pHs from 3.0 to 7.4.

[0058]FIG. 2 shows the data for TEER of permeability enhancers.

[0059]FIG. 3 shows the cell viabilities of candidate PYY formulations.

[0060]FIG. 4 shows the cytotoxic effects of candidate formulations. InFIGS. 2-4

[0061] EN1=PBS pH 5.0

[0062] EN2=L-Arginine (10% w/v)

[0063] EN3=Poly-L-Arginine (0.5% w/v)

[0064] EN4=Gamma-Cyclodextrin (1% w/v)

[0065] EN5=Alpha-Cyclodextrin (5% w/v)

[0066] EN6=Methyl-Beta-Cyclodextrin (3% w/v)

[0067] EN7=n-Capric Acid Sodium (0.075% w/v)

[0068] EN8=Chitosan (0.5% w/v)

[0069] EN9=L-Alpha-phosphafidilcholine didecanyl (3.5% w/v)

[0070] EN10=S-Nitroso-N-acetylpenicillamine, (0.02% w/v)

[0071] EN11=Palmotoyl-DL-Carnitine (0.5% w/v)

[0072] EN12=Pluronic-127 (0.3% w/v)

[0073] EN13=Sodium Nitroprusside (0.3% w/v)

[0074] EN14=Sodium Glycocholate (1% w/v)

[0075]FIG. 5 shows the synergistic contributions of the variouscomponents on drug permeation. In FIG. 5 EN1 is DDPC, EN2 ismethyl-β-cyclodextrin, and EX1 is EDTA.

[0076]FIG. 6 shows the PYY3-36 in the plasma of rats, the squarerepresent a dose of 4.1 μg/kg, the triangle represents a dose of 41μg/kg, and the circle represent a dose of 205 μg/kg.

[0077]FIG. 7 shows dose linearity following intranasal administrationPYY3-36 in rats as Cmax-Cbas pg/mL v. dose as μg/kg.

[0078]FIG. 8 shows dose linearity following intranasal administration ofPYY3-36 in rats as AUC v. dose as μg/kg.

[0079]FIG. 9 shows the average plasma concentration of PYY v. time inminutes in three human volunteers who were each administered 20 μg ofPYY(3-36) intranasally.

[0080]FIG. 10 shows the average plasma concentration of PYY v. time inminutes in three human volunteers who were each administered 50 μg ofPYY(3-36) intranasally.

[0081]FIG. 11 shows the average plasma concentration of PYY v. time inminutes in three human volunteers who were each administered 100 μg ofPYY(3-36) intranasally.

[0082]FIG. 12 shows the average plasma concentration of PYY v. time inminutes in three human volunteers who were each administered 150 μg ofPYY3-36 intranasally.

[0083]FIG. 13 shows the average plasma concentration of PYY v. time inminutes in three human volunteers who were each administered 200 μg ofPYY(3-36) intranasally.

[0084]FIG. 14 shows PYY plasma concentration as pmol/L v. time for fivegroups of healthy human volunteers who received intranasal PYY(3-36).The doses were 200 μg, 150 μg, 100 μg, 50 μg and 20 μg of PYY3-36.

[0085]FIG. 15 shows the dose linearity Cmax of PYY in pg/mL vs. dose ofPYY(3-36) administered to human volunteers.

[0086]FIG. 16 shows the dose linearity PYY mean AUC in pg/mL vs. dose ofPYY(3-36) administered to human volunteers.

[0087]FIG. 17 shows the visual analog scale (VAS) vs. dose of PYY(3-36)administered to the human volunteers. The question was: “How hungry areyou?” The lower the score the less hungry an individual was on a 100point scale.

[0088]FIG. 18 shows the visual analog scale (VAS) vs. dose of PYY(3-36)administered to the human volunteers. The question was: “How much couldyou eat?” The lower the score the less hungry an individual was on a 100point scale.

[0089]FIG. 19 shows the visual analog scale (VAS) vs. dose of PYY(3-36)administered to the human volunteers. The question was: “How full do youfeel?” The lower the score the less full an individual was on a 100point scale.

[0090]FIG. 20 shows the percent permeation of PYY(3-36) containingendotoxin vs. endotoxin-free PYY(3-36).

DETAILED DESCRIPTION OF THE INVENTION

[0091] As noted above, the present invention provides improved methodsand compositions for mucosal delivery of Y2 receptor-binding peptide tomammalian subjects for treatment or prevention of a variety of diseasesand conditions. Examples of appropriate mammalian subjects for treatmentand prophylaxis according to the methods of the invention include, butare not restricted to, humans and non-human primates, livestock species,such as horses, cattle, sheep, and goats, and research and domesticspecies, including dogs, cats, mice, rats, guinea pigs, and rabbits.

[0092] In order to provide better understanding of the presentinvention, the following definitions are provided:

Y2 Receptor-Binding Peptides

[0093] The Y2 receptor-binding peptides used in the mucosal formulationsof the present invention include the pancreatic polypeptide family.” asused herein, is comprised of three naturally occurring bioactive peptidefamilies, PP, NPY, and PYY. Examples of Y2 receptor-binding peptides andtheir uses are described in U.S. Pat. No. 5,026,685; U.S. Pat. No.5,574,010; U.S. Pat. No. 5,604,203; U.S. Pat. No. 5,696,093; U.S. Pat.No. 6,046,167; Gehlert et.al., Proc Soc Exp Biol Med 218:7-22 (1998);Sheikh et al. Am J Physiol, 261:701-15(1991); Fournier et al., MolPharmacol 45:93-101 (1994); Kirby et al., J Med Chem 38:4579-4586(1995); Rist et al., Eur J Biochem 247: 1019-1028 (1997); Kirby etal.,J. Med Chem 36:3802-3808 (1993); Grundemar et al., RegulatoryPeptides 62: 131-136 (1996); U.S. Pat. No. 5,696,093 (examples of PYYagonists), U.S. Pat. No. 6,046,167. According to the present invention aY2 receptor-binding peptide includes the free bases, acid addition saltsor metal salts, such as potassium or sodium salts or the peptides Y2receptor-binding peptides that have been modified by such processes asamidation, glycosylation, acylation, sulfation, phosphorylation,acetylation and cyclization, (U.S. Pat. No. 6,093,692; and U.S. Pat. No.6,225,445 and pegylation.

Peptide YY Agonists

[0094] As used herein, “PYY” refers to PYY(1-36) in native-sequence orin variant form, as well as derivatives, fragments, and analogs of PYYfrom any source, whether natural, synthetic, or recombinant. The PYYmust be comprised at least the last 15 amino acid residues or analogouesthereof of the PYY sequence,PYY(22-36) (SEQ ID NO: 3). Other PYYpeptides, which may be used are PYY(1-36) (SEQ ID NO: 1) PYY(3-36) SEQID NO: 2) PYY(4-36)(SEQ ID NO:4) PYY(5-36) (SEQ ID NO: 5), PYY(6-36)(SEQ ID NO:6), PYY(7-36) (SEQ ID NO:7) PYY(8-36) (SEQ ID NO: 8), PYY9-36(SEQ ID NO: 9) PYY(10-36) (SEQ ID NO: 10), PYY(11-36) (SEQ ID NO: 11),PYY(12-36) (SEQ ID NO: 12), PYY(13-36) (SEQ ID NO: 13), PYY(14-36) (SEQID NO: 14), PYY(15-36) (SEQ ID NO: 15), PYY(16-36) (SEQ ID NO: 16),PYY(17-36) (SEQ ID NO: 17), PYY(18-36) (SEQ ID NO: 18), PYY(19-36) (SEQID NO: 19), PYY(20-36) (SEQ ID NO: 20) and PYY(21-36) (SEQ ID NO: 21).These peptides typically bind to the Y receptors in the brain andelsewhere, especially the Y2 and/or Y5 receptors. Typically thesepeptides are synthesized in endotoxin-free or pyrogen-free formsalthough this is not always necessary.

[0095] Other PYY peptides include those PYY peptides in whichconservative amino acid residue changes have beem made, for example,site specific mutation of a PYY peptide including [Asp¹⁵] PYY(15-36)(SEQ ID NO: 90), [Thr¹³] PYY(13-36) (SEQ ID NO: 91), [Val¹²]PYY(12-36)(SEQ ID NO: 92), [Glu¹¹] PYY(11-36) (SEQ ID NO: 93), [Asp¹⁰]PYY(10-36) (SEQ ID NO: 94), [Val⁷] PYY(7-36) (SEQ ID NO: 95), [Asp⁶]PYY(6-36) (SEQ ID NO: 96), [Gln⁴] PYY(4-36) (SEQ ID NO: 97), [Arg⁴]PYY(4-36) (SEQ ID NO: 98), [Asn⁴] PYY(4-36) (SEQ ID NO: 99), [Val³]PYY(3-36) (SEQ ID NO: 100) and [Leu³] PYY(3-36) (SEQ ID NO: 101). OtherPYY peptides include those peptides in which at least two conservativeamino acid residue changes have been made including [Asp¹⁰, Asp¹⁵]PYY(10-36) (SEQ ID NO: 102), [Asp⁶, Thr13] PYY(6-36) (SEQ ID NO: 103),[Asn⁴, Asp¹⁵] PYY(4-36) (SEQ ID NO: 104) and [Leu³, Asp¹⁰] PYY(3-36)(SEQ ID NO: 105. Also included are analogues of a PYY for example thosedisclosed in U.S. Pat. Nos. 5,604,203 and 5,574,010; Balasubramaniam, etal., Peptide Research 1: 32 (1988); Japanese Patent Application2,225,497 (1990); Balasubramaniam, et al., Peptides 14: 1011, 1993;Grandt, et at., Reg. Peptides 51: 151, (1994); PCT InternationalApplication 94/03380, U.S. Pat. Nos. 5,604,203 and 5,574,010. Thesepeptides typically bind to the Y receptors in the brain and elsewhere,especially the Y2 and/or Y5 receptors. Typically these peptides aresynthesized in endotoxin-free or pyrogen-free forms although this is notalways necessary.

[0096] PYY agonists include rat PYY (SEQ ID NO: 72) and the aminoterminus truncated forms corresponding to the human, pig PYY (SEQ ID NO:73) and the amino terminus truncated forms corresponding to the humanand guinea pig PYY (SEQ ID NO: 74) and the amino terminus truncatedforms corresponding to the human.

[0097] According to the present invention a PYY peptide also includesthe free bases, acid addition salts or metal salts, such as potassium orsodium salts of the peptides, and PYY peptides that have been modifiedby such processes as amidation, glycosylation, acylation, sulfation,phosphorylation, acetylation, cyclization and other well known covalentmodification methods. These peptides typically bind to the Y receptorsin the brain and elsewhere, especially the Y2 and/or Y5 receptors.Typically these peptides are synthesized in endotoxin-free orpyrogen-free forms although this is not always necessary.

Neuropeptide Y Agonists

[0098] NPY is another Y2 receptor-binding peptide. NPY peptides includefull-length NPY(1-36) (SEQ ID NO: 22) as well as well as fragments ofNPY(1-36), which have been truncated at the amino terminus. To beeffective in binding the Y2 receptor, the NPY agonist should have atleast the lastl 1 amino acid residues at the carboxyl terminus, i.e., becomprised of NPY(26-36) (SEQ ID NO: 23). Other examples of NPY agoniststhat bind to the Y2 receptor are NPY(3-36) (SEQ ID NO: 24), NPY(4-36)(SEQ ID NO: 25), NPY(5-36) (SEQ ID NO: 26), NPY(6-36) (SEQ ID NO: 27),NPY(7-36) (SEQ ID NO: 28), NPY(8-36) (SEQ ID NO: 29), NPY(9-36) (SEQ IDNO: 30), NPY(10-36) (SEQ ID NO: 31), NPY(11-36) (SEQ ID NO: 32),NPY(12-36) (SEQ ID NO: 33), NPY(13-36) (SEQ ID NO: 34), NPY(14-36) (SEQID NO: 35), NPY(15-36) (SEQ ID NO: 36), NPY(16-36) (SEQ ID NO: 37),NPY(17-36) (SEQ ID NO: 38), NPY(18-36) (SEQ ID NO: 39), NPY(19-36) (SEQID NO: 40), NPY(20-36) (SEQ ID NO: 41), NPY(21-36) (SEQ ID NO: 42),NPY(22-36) (SEQ ID NO: 43), NPY(23-36) (SEQ ID NO: 44), NPY(24-36) (SEQID NO: 45) and NPY(25-36) (SEQ ID NO: 46).

[0099] Other NPY agonists include rat NPY (SEQ ID NO: 75) and the aminoterminus truncated forms from NPY(3-36) to NPY(26-36) as in the humanform, rabbit NPY (SEQ ID NO: 76) and the amino terminus truncated formsfrom NPY(3-36) to NPY(26-36) as in the human form, dog NPY (SEQ ID NO:77) and the amino terminus truncated forms NPY(3-36) to NPY(26-36) as inthe human form, pig NPY (SEQ ID NO: 78) and the amino terminus truncatedforms from NPY(3-36) to NPY(26-36) as in the human form, cow NPY (SEQ IDNO: 79) and the amino terminus truncated forms from NPY(3-36) toNPY26-36 as in the human form, sheep NPY (SEQ ID NO:80) and the aminoterminus truncated forms from NPY(3-36) to NPY(26-36) as in the humanform and guinea pig (SEQ 81) and the amino terminus truncated forms fromNPY(3-36) to NPY(26-36) as in the human form.

[0100] According to the present invention a NPY peptide also includesthe free bases, acid additoin salts or metal salts, such as potassium orsodium salts of the peptides, and NPY peptides that have been modifiedby such processes as amidation, glycosylation, acylation, sulfation,phosphorylation, acetylation, cyclization and other known covalentmodification methods. These peptides typically bind to the Y receptorsin the brain and elsewhere, especially the Y2 and/or Y5 receptors.Typically these peptides are synthesized in endotoxin-free orpyrogen-free forms although this is not always necessary.

Pancreatic Peptide

[0101] Pancreatic Peptide (PP) and PP agonist also bind to the Y2receptor. Examples of the PP agonists are the full-length PP(1-36) (SEQID NO: 47) and a number of PP fragments, which are truncated at theamino-terminus. To bind to the Y2 receptor the PP agonist must have thelast 11 amino acid residues at the carboxyl-terminus, PP(26-36), (SEQ IDNO: 48). Examples of other PP, which bind to the Y2 receptor, arePP(3-36) (SEQ ID NO: 49), PP(4-36) (SEQ ID NO: 50), PP(5-36) (SEQ ID NO:51), PP(6-36) (SEQ ID NO: 52), PP(7-36) (SEQ ID NO: 53), PP(8-36) (SEQID NO: 54), PP(9-36) (SEQ ID NO: 55), PP(10-36) (SEQ ID NO: 56),PP(11-36) (SEQ ID NO: 57), PP(12-36) (SEQ ID NO: 58), PP(13-36) (SEQ IDNO: 59), PP(14-36) (SEQ ID NO: 60), PP(15-36) (SEQ ID NO: 61), PP(16-36)(SEQ ID NO: 62), PP(17-36) (SEQ ID NO: 63), PP(18-36) (SEQ ID NO: 64),PP(19-36) (SEQ ID NO: 65), PP(20-36) (SEQ ID NO: 66), PP(21-36) (SEQ IDNO: 67), PP(22-36) (SEQ ID NO: 68), PP(23-36) (SEQ ID NO: 69), PP(24-36)(SEQ ID NO: 70) and PP(25-36) (SEQ ID NO: 71).

[0102] Other PP agonists include sheep PP (SEQ ID NO: 82) and the aminoterminus truncated forms from PP(3-36) to PP(26-36) as in the humanform, pig PP (SEQ ID NO: 83) and the amino terminus truncated forms fromPP(3-36) to PP(26-36) as in the human form, dog PP (SEQ ID NO: 84) andthe amino terminus truncated forms PP(3-36) to PP(26-36) as in the humanform, cat PP (SEQ ID NO: 85) and the amino terminus truncated forms fromPP(3-36) to PP(26-36) as in the human form, cow PP (SEQ ID NO: 86) andthe amino terminus truncated forms from PP(3-36) to PP(26-36) as in thehuman form, rat PP (SEQ ID NO:87) and the amino terminus truncated formsfrom PP(3-36) to PP(26-36) as in the human form, mouse (SEQ 88) and theamino terminus truncated forms from PP(3-36) to PP(26-36) as in thehuman form, and guinea pig PP (SEQ ID NO: 89).

[0103] According to the present invention a PP peptide also includes thefree bases, acid additoin salts or metal salts, such as potassium orsodium salts of the peptides, and PP peptides that have been modified bysuch processes as amidation, glycosylation, acylation, sulfation,phosphorylation, acetylation, cyclization, and other known covalentmodification methods. These peptides typically bind to the Y receptorsin the brain and elsewhere, especially the Y2 and/or Y5 receptors.Typically these peptides are synthesized in endotoxin-free orpyrogen-free forms although this is not always necessary. MucosalDelivery Enhancing Agents

[0104] “Mucosal delivery enhancing agents” are defined as chemicals andother excipients that, when added to a formulation comprising water,salts and/or common buffers and Y2 receptor-binding peptide (the controlformulation) produce a formulation that produces a significant increasein transport of Y2 receptor-binding peptide across a mucosa as measuredby the maximum blood, serum, or cerebral spinal fluid concentration(C_(max)) or by the area under the curve, AUC, in a plot ofconcentration versus time. A mucosa includes the nasal, oral,intestional, buccal, bronchopulmonary, vaginal, and rectal mucosalsurfaces and in fact includes all mucus-secreting membranes lining allbody cavities or passages that communicate with the exterior. Mucosaldelivery enhancing agents are sometimes called carriers.

[0105] Endotoxin-Free Formulation

[0106] “Endotoxin-free formulation” means a formulation which contains aY2-receptor-binding peptide and one or more mucosal delivery enhancingagents that is substantially free of endotoxins and/or related pyrogenicsubstances. Endotoxins include toxins that are confined inside amicroorganism and are released only when the microorganisms are brokendown or die. Pyrogenic substances include fever-inducing, thermostablesubstances (glycoproteins) from the outer membrane of bacteria and othermicroorganisms. Both of these substances can cause fever, hypotensionand shock if administered to humans. Producing formulations that areendotoxin-free can require special equipment, expert artisians, and canbe significantly more expensive than making formulations that are notendotoxin-free. Because intravenous administration of NPY or PYYsimultaneously with infusion of endotoxin in rodents has been shown toprevent the hypotension and even death associated with theadministration of endotoxin alone (U.S. Pat. No. 4,839,343), producingendotoxin-free formulations of these therapeutic agents would not beexpected to be necessary for non-parental (non-injected) administration.

[0107] Non-infused Administration

[0108] “Non-infused administration” means any method of delivery thatdoes not involve an injection directly into an artery or vein, a methodwhich forces or drives (typically a fluid) into something and especiallyto introduce into a body part by means of a needle, syringe or otherinvasive method. Non-infused administration includes subcutaneousinjection, intramuscular injection, intraparitoneal injection and thenon-injection methods of delivery to a mucosa.

[0109] Treatment and Prevention of Obesity

[0110] As noted above, the instant invention provides improved anduseful methods and compositions for nasal mucosal delivery of a Y2receptor-binding peptide to prevent and treat obesity in mammaliansubjects. As used herein, prevention and treatment of obesity meanprevention of the onset or lowering the incidence or severity ofclinical obesity by reducing food intake during meals and/or reducingbody weight during administration or maintaining a reduced body weightfollowing weight loss or before weight gain has occurred.

[0111] The instant invention provides improved and useful methods andcompositions for nasal mucosal delivery of Y2 receptor-binding peptideto regions of the brain, for example, the hypothalamus or theproopiomelanocortin (POMC) and NPY arcuate neurons, to prevent and treatobesity in mammalian subjects. The Y2 receptor-binding peptide can alsobe administered in conjunction with a Y1 receptor antagonist such asdihyropyridine.

[0112] Methods and Compositions of Delivery

[0113] Improved methods and compositions for mucosal administration ofY2 receptor-binding peptide to mammalian subjects optimize Y2receptor-binding peptide dosing schedules. The present inventionprovides mucosal delivery of Y2 receptor-binding peptide formulated withone or more mucosal delivery-enhancing agents wherein Y2receptor-binding peptide dosage release is substantially normalizedand/or sustained for an effective delivery period of Y2 receptor-bindingpeptide release ranges from approximately 0.1 to 2.0 hours; 0.4 to 1.5hours; 0.7 to 1.5 hours; or 0.8 to 1.0 hours; following mucosaladministration. The sustained release of Y2 receptor-binding peptideachieved may be facilitated by repeated administration of exogenous Y2receptor-binding peptide utilizing methods and compositions of thepresent invention.

[0114] Compositions and Methods of Sustained Release

[0115] Improved compositions and methods for mucosal administration ofY2 receptor-binding peptide to mammalian subjects optimize Y2receptor-binding peptide dosing schedules. The present inventionprovides improved mucosal (e.g., nasal) delivery of a formulationcomprising Y2 receptor-binding peptide in combination with one or moremucosal delivery-enhancing agents and an optional sustainedrelease-enhancing agent or agents. Mucosal delivery-enhancing agents ofthe present invention yield an effective increase in delivery, e.g., anincrease in the maximal plasma concentration (C_(max)) to enhance thetherapeutic activity of mucosally-administered Y2 receptor-bindingpeptide. A second factor affecting therapeutic activity of Y2receptor-binding peptide in the blood plasma and CNS is residence time(RT). Sustained release-enhancing agents, in combination with intranasaldelivery-enhancing agents, increase C_(max) and increase residence time(RT) of Y2 receptor-binding peptide. Polymeric delivery vehicles andother agents and methods of the present invention that yield sustainedrelease-enhancing formulations, for example, polyethylene glycol (PEG),are disclosed herein. The present invention provides an improved Y2receptor-binding peptide delivery method and dosage form for treatmentof symptoms related to obesity, colon cancer, pancreatic cancer, orbreast cancer in mammalian subjects.

[0116] Within the mucosal delivery formulations and methods of theinvention, the Y2 receptor-binding peptide is frequently combined orcoordinately administered with a suitable carrier or vehicle for mucosaldelivery. As used herein, the term “carrier” means a pharmaceuticallyacceptable solid or liquid filler, diluent or encapsulating material. Awater-containing liquid carrier can contain pharmaceutically acceptableadditives such as acidifying agents, alkalizing agents, antimicrobialpreservatives, antioxidants, buffering agents, chelating agents,complexing agents, solubilizing agents, humectants, solvents, suspendingand/or viscosity-increasing agents, tonicity agents, wetting agents orother biocompatible materials. A tabulation of ingredients listed by theabove categories, can be found in the US. Pharmacopeia NationalFormulary, 1857-1859, (1990). Some examples of the materials which canserve as pharmaceutically acceptable carriers are sugars, such aslactose, glucose and sucrose; starches such as corn starch and potatostarch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; powdered tragacanth;malt; gelatin; talc; excipients such as cocoa butter and suppositorywaxes; oils such as peanut oil, cottonseed oil, safflower oil, sesameoil, olive oil, corn oil and soybean oil; glycols, such as propyleneglycol; polyols such as glycerin, sorbitol, mannitol and polyethyleneglycol; esters such as ethyl oleate and ethyl laurate; agar; bufferingagents such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen free water; isotonic saline; Ringer's solution, ethyl alcoholand phosphate buffer solutions, as well as other non toxic compatiblesubstances used in pharmaceutical formulations. Wetting agents,emulsifiers and lubricants such as sodium lauryl sulfate and magnesiumstearate, as well as coloring agents, release agents, coating agents,sweetening, flavoring and perfuming agents, preservatives andantioxidants can also be present in the compositions, according to thedesires of the formulator. Examples of pharmaceutically acceptableantioxidants include water soluble antioxidants such as ascorbic acid,cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodiumsulfite and the like; oil-soluble antioxidants such as ascorbylpalmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene(BHT), lecithin, propyl gallate, alpha-tocopherol and the like; andmetal-chelating agents such as citric acid, ethylenediamine tetraaceticacid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like. Theamount of active ingredient that can be combined with the carriermaterials to produce a single dosage form will vary depending upon theparticular mode of administration.

[0117] Within the mucosal delivery compositions and methods of theinvention, various delivery-enhancing agents are employed which enhancedelivery of Y2 receptor-binding peptide into or across a mucosalsurface. In this regard, delivery of Y2 receptor-binding peptide acrossthe mucosal epithelium can occur “transcellularly” or “paracellularly”.The extent to which these pathways contribute to the overall flux andbioavailability of the Y2 receptor-binding peptide depends upon theenvironment of the mucosa, the physico-chemical properties the activeagent, and on the properties of the mucosal epithelium. Paracellulartransport involves only passive diffusion, whereas transcellulartransport can occur by passive, facilitated or active processes.Generally, hydrophilic, passively transported, polar solutes diffusethrough the paracellular route, while more lipophilic solutes use thetranscellular route. Absorption and bioavailability (e.g., as reflectedby a permeability coefficient or physiological assay), for diverse,passively and actively absorbed solutes, can be readily evaluated, interms of both paracellular and transcellular delivery components, forany selected Y2 receptor-binding peptide within the invention. Forpassively absorbed drugs, the relative contribution of paracellular andtranscellular pathways to drug transport depends upon the pKa, partitioncoefficient, molecular radius and charge of the drug, the pH of theluminal environment in which the drug is delivered, and the area of theabsorbing surface. The paracellular route represents a relatively smallfraction of accessible surface area of the nasal mucosal epithelium. Ingeneral terms, it has been reported that cell membranes occupy a mucosalsurface area that is a thousand times greater than the area occupied bythe paracellular spaces. Thus, the smaller accessible area, and thesize- and charge-based discrimination against macromolecular permeationwould suggest that the paracellular route would be a generally lessfavorable route than transcellular delivery for drug transport.Surprisingly, the methods and compositions of the invention provide forsignificantly enhanced transport of biotherapeutics into and acrossmucosal epithelia via the paracellular route. Therefore, the methods andcompositions of the invention successfully target both paracellular andtranscellular routes, alternatively or within a single method orcomposition.

[0118] As used herein, “mucosal delivery-enhancing agents” includeagents which enhance the release or solubility (e.g., from a formulationdelivery vehicle), diffusion rate, penetration capacity and timing,uptake, residence time, stability, effective half-life, peak orsustained concentration levels, clearance and other desired mucosaldelivery characteristics (e.g., as measured at the site of delivery, orat a selected target site of activity such as the bloodstream or centralnervous system) of Y2 receptor-binding peptide or other biologicallyactive compound(s). Enhancement of mucosal delivery can thus occur byany of a variety of mechanisms, for example by increasing the diffusion,transport, persistence or stability of Y2 receptor-binding peptide,increasing membrane fluidity, modulating the availability or action ofcalcium and other ions that regulate intracellular or paracellularpermeation, solubilizing mucosal membrane components (e.g., lipids),changing non-protein and protein sulfhydryl levels in mucosal tissues,increasing water flux across the mucosal surface, modulating epithelialjunctional physiology, reducing the viscosity of mucus overlying themucosal epithelium, reducing mucociliary clearance rates, and othermechanisms.

[0119] As used herein, a “mucosally effective amount of Y2receptor-binding peptide” contemplates effective mucosal delivery of Y2receptor-binding peptide to a target site for drug activity in thesubject that may involve a variety of delivery or transfer routes. Forexample, a given active agent may find its way through clearancesbetween cells of the mucosa and reach an adjacent vascular wall, whileby another route the agent may, either passively or actively, be takenup into mucosal cells to act within the cells or be discharged ortransported out of the cells to reach a secondary target site, such asthe systemic circulation. The methods and compositions of the inventionmay promote the translocation of active agents along one or more suchalternate routes, or may act directly on the mucosal tissue or proximalvascular tissue to promote absorption or penetration of the activeagent(s). The promotion of absorption or penetration in this context isnot limited to these mechanisms.

[0120] As used herein “peak concentration (C_(max)) of Y2receptor-binding peptide in a blood plasma”, “area under concentrationvs. time curve (AUC) of Y2 receptor-binding peptide in a blood plasma”,“time to maximal plasma concentration (t_(max)) of Y2 receptor-bindingpeptide in a blood plasma” are pharmacokinetic parameters known to oneskilled in the art. Laursen et al., Eur. J. Endocrinology, 135: 309-315,1996. The “concentration vs. time curve” measures the concentration ofY2 receptor-binding peptide in a blood serum of a subject vs. time afteradministration of a dosage of Y2 receptor-binding peptide to the subjecteither by intranasal, intramuscular, subcutaneous, or other parenteralroute of administration. “C_(max)” is the maximum concentration of Y2receptor-binding peptide in the blood serum of a subject following asingle dosage of Y2 receptor-binding peptide to the subject. “t_(max)”is the time to reach maximum concentration of Y2 receptor-bindingpeptide in a blood serum of a subject following administration of asingle dosage of Y2 receptor-binding peptide to the subject.

[0121] As used herein, “area under concentration vs. time curve (AUC) ofY2 receptor-binding peptide in a blood plasma” is calculated accordingto the linear trapezoidal rule and with addition of the residual areas.A decrease of 23% or an increase of 30% between two dosages would bedetected with a probability of 90% (type II error β=10%). The “deliveryrate” or “rate of absorption” is estimated by comparison of the time(t_(max)) to reach the maximum concentration (C_(max)). Both C_(max) andt_(max) are analyzed using non-parametric methods. Comparisons of thepharmacokinetics of intramuscular, subcutaneous, intravenous andintranasal Y2 receptor-binding peptide administrations were performed byanalysis of variance (ANOVA). For pair wise comparisons aBonferroni-Holmes sequential procedure was used to evaluatesignificance. The dose-response relationship between the three nasaldoses was estimated by regression analysis. P<0.05 was consideredsignificant. Results are given as mean values+/−SEM.

[0122] While the mechanism of absorption promotion may vary withdifferent mucosal delivery-enhancing agents of the invention, usefulreagents in this context will not substantially adversely affect themucosal tissue and will be selected according to the physicochemicalcharacteristics of the particular Y2 receptor-binding peptide or otheractive or delivery-enhancing agent. In this context, delivery-enhancingagents that increase penetration or permeability of mucosal tissues willoften result in some alteration of the protective permeability barrierof the mucosa. For such delivery-enhancing agents to be of value withinthe invention, it is generally desired that any significant changes inpermeability of the mucosa be reversible within a time frame appropriateto the desired duration of drug delivery. Furthermore, there should beno substantial, cumulative toxicity, nor any permanent deleteriouschanges induced in the barrier properties of the mucosa with long-termuse.

[0123] Within certain aspects of the invention, absorption-promotingagents for coordinate administration or combinatorial formulation withY2 receptor-binding peptide of the invention are selected from smallhydrophilic molecules, including but not limited to, dimethyl sulfoxide(DMSO), dimethylformamide, ethanol, propylene glycol, and the2-pyrrolidones. Alternatively, long-chain amphipathic molecules, forexample, deacylmethyl sulfoxide, azone, sodium laurylsulfate, oleicacid, and the bile salts, may be employed toenhance mucosal penetrationof the Y2 receptor-binding peptide. In additional aspects, surfactants(e.g., polysorbates) are employed as adjunct compounds, processingagents, or formulation additives to enhance intranasal delivery of theY2 receptor-binding peptide. Agents such as DMSO, polyethylene glycol,and ethanol can, if present in sufficiently high concentrations indelivery environment (e.g., by pre-administration or incorporation in atherapeutic formulation), enter the aqueous phase of the mucosa andalter its solubilizing properties, thereby enhancing the partitioning ofthe Y2 receptor-binding peptide from the vehicle into the mucosa.

[0124] Additional mucosal delivery-enhancing agents that are usefulwithin the coordinate administration and processing methods andcombinatorial formulations of the invention include, but are not limitedto, mixed micelles; enamines; nitric oxide donors (e.g.,S-nitroso-N-acetyl-DL-penicillamine, NOR1, NOR4—which are preferablyco-administered with an NO scavenger such as carboxy-PITO or doclofenacsodium); sodium salicylate; glycerol esters of acetoacetic acid (e.g.,glyceryl-1,3-diacetoacetate or1,2-isopropylideneglycerine-3-acetoacetate); and other release-diffusionor intra- or trans-epithelial penetration-promoting agents that arephysiologically compatible for mucosal delivery. Otherabsorption-promoting agents are selected from a variety of carriers,bases and excipients that enhance mucosal delivery, stability, activityor trans-epithelial penetration of the Y2 receptor-binding peptide.These include, inter alia, cyclodextrins and β-cyclodextrin derivatives(e.g., 2-hydroxypropyl-β-cyclodextrin andheptakis(2,6-di-O-methyl-β-cyclodextrin). These compounds, optionallyconjugated with one or more of the active ingredients and furtheroptionally formulated in an oleaginous base, enhance bioavailability inthe mucosal formulations of the invention. Yet additionalabsorption-enhancing agents adapted for mucosal delivery includemedium-chain fatty acids, including mono- and diglycerides (e.g., sodiumcaprate—extracts of coconut oil, Capmul), and triglycerides (e.g.,amylodextrin, Estaram 299, Miglyol 810).

[0125] The mucosal therapeutic and prophylactic compositions of thepresent invention may be supplemented with any suitablepenetration-promoting agent that facilitates absorption, diffusion, orpenetration of Y2 receptor-binding peptide across mucosal barriers. Thepenetration promoter may be any promoter that is pharmaceuticallyacceptable. Thus, in more detailed aspects of the invention compositionsare provided that incorporate one or more penetration-promoting agentsselected from sodium salicylate and salicylic acid derivatives (acetylsalicylate, choline salicylate, salicylamide, etc.); amino acids andsalts thereof (e.g. monoaminocarboxlic acids such as glycine, alanine,phenylalanine, proline, hydroxyproline, etc.; hydroxyamino acids such asserine; acidic amino acids such as aspartic acid, glutamic acid, etc;and basic amino acids such as lysine etc-inclusive of their alkali metalor alkaline earth metal salts); and N-acetylamino acids(N-acetylalanine, N-acetylphenylalanine, N-acetylserine,N-acetylglycine, N-acetyllysine, N-acetylglutamic acid, N-acetylproline,N-acetylhydroxyproline, etc.) and their salts (alkali metal salts andalkaline earth metal salts). Also provided as penetration-promotingagents within the methods and compositions of the invention aresubstances which are generally used as emulsifiers (e.g. sodium oleylphosphate, sodium lauryl phosphate, sodium lauryl sulfate, sodiummyristyl sulfate, polyoxyethylene alkyl ethers, polyoxyethylene alkylesters, etc.), caproic acid, lactic acid, malic acid and citric acid andalkali metal salts thereof, pyrrolidonecarboxylic acids,alkylpyrrolidonecarboxylic acid esters, N-alkylpyrrolidones, prolineacyl esters, and the like.

[0126] Within various aspects of the invention, improved nasal mucosaldelivery formulations and methods are provided that allow delivery of Y2receptor-binding peptide and other therapeutic agents within theinvention across mucosal barriers between administration and selectedtarget sites. Certain formulations are specifically adapted for aselected target cell, tissue or organ, or even a particular diseasestate. In other aspects, formulations and methods provide for efficient,selective endo- or transcytosis of Y2 receptor-binding peptidespecifically routed along a defined intracellular or intercellularpathway. Typically, the Y2 receptor-binding peptide is efficientlyloaded at effective concentration levels in a carrier or other deliveryvehicle, and is delivered and maintained in a stabilized form, e.g., atthe nasal mucosa and/or during passage through intracellularcompartments and membranes to a remote target site for drug action(e.g., the blood stream or a defined tissue, organ, or extracellularcompartment). The Y2 receptor-binding peptide may be provided in adelivery vehicle or otherwise modified (e.g., in the form of a prodrug),wherein release or activation of the Y2 receptor-binding peptide istriggered by a physiological stimulus (e.g. pH change, lysosomalenzymes, etc.) Often, the Y2 receptor-binding peptide ispharmacologically inactive until it reaches its target site foractivity. In most cases, the Y2 receptor-binding peptide and otherformulation components are non-toxic and non-immunogenic. In thiscontext, carriers and other formulation components are generallyselected for their ability to be rapidly degraded and excreted underphysiological conditions. At the same time, formulations are chemicallyand physically stable in dosage form for effective storage.

[0127] Peptide and Protein Analogs and Mimetics

[0128] Included within the definition of biologically active peptidesand proteins for use within the invention are natural or synthetic,therapeutically or prophylactically active, peptides (comprised of twoor more covalently linked amino acids), proteins, peptide or proteinfragments, peptide or protein analogs, and chemically modifiedderivatives or salts of active peptides or proteins. A wide variety ofuseful analogs and mimetics of Y2 receptor-binding peptide arecontemplated for use within the invention and can be produced and testedfor biological activity according to known methods. Often, the peptidesor proteins of Y2 receptor-binding peptide or other biologically activepeptides or proteins for use within the invention are muteins that arereadily obtainable by partial substitution, addition, or deletion ofamino acids within a naturally occurring or native (e.g., wild-type,naturally occurring mutant, or allelic variant) peptide or proteinsequence. Additionally, biologically active fragments of native peptidesor proteins are included. Such mutant derivatives and fragmentssubstantially retain the desired biological activity of the nativepeptide or proteins. In the case of peptides or proteins havingcarbohydrate chains, biologically active variants marked by alterationsin these carbohydrate species are also included within the invention.

[0129] As used herein, the term “conservative amino acid substitution”refers to the general interchangeability of amino acid residues havingsimilar side chains. For example, a commonly interchangeable group ofamino acids having aliphatic side chains is alanine, valine, leucine,and isoleucine; a group of amino acids having aliphatic-hydroxyl sidechains is serine and threonine; a group of amino acids havingamide-containing side chains is asparagine and glutamine; a group ofamino acids having aromatic side chains is phenylalanine, tyrosine, andtryptophan; a group of amino acids having basic side chains is lysine,arginine, and histidine; and a group of amino acids havingsulfur-containing side chains is cysteine and methionine. Examples ofconservative substitutions include the substitution of a non-polar(hydrophobic) residue such as isoleucine, valine, leucine or methioninefor another. Likewise, the present invention contemplates thesubstitution of a polar (hydrophilic) residue such as between arginineand lysine, between glutamine and asparagine, and between threonine andserine. Additionally, the substitution of a basic residue such aslysine, arginine or histidine for another or the substitution of anacidic residue such as aspartic acid or glutamic acid for another isalso contemplated. Exemplary conservative amino acids substitutiongroups are: valine-leucine-isoleucine, phenylalanine-tyrosine,lysine-arginine, alanine-valine, and asparagine-glutamine. By aligning apeptide or protein analog optimally with a corresponding native peptideor protein, and by using appropriate assays, e.g., adhesion protein orreceptor binding assays, to determine a selected biological activity,one can readily identify operable peptide and protein analogs for usewithin the methods and compositions of the invention. Operable peptideand protein analogs are typically specifically immunoreactive withantibodies raised to the corresponding native peptide or protein.

[0130] An approach for stabilizing solid protein formulations of theinvention is to increase the physical stability of purified, e.g.,lyophilized, protein. This will inhibit aggregation via hydrophobicinteractions as well as via covalent pathways that may increase asproteins unfold. Stabilizing formulations in this context often includepolymer-based formulations, for example a biodegradable hydrogelformulation/delivery system. As noted above, the critical role of waterin protein structure, function, and stability is well known. Typically,proteins are relatively stable in the solid state with bulk waterremoved. However, solid therapeutic protein formulations may becomehydrated upon storage at elevated humidities or during delivery from asustained release composition or device. The stability of proteinsgenerally drops with increasing hydration. Water can also play asignificant role in solid protein aggregation, for example, byincreasing protein flexibility resulting in enhanced accessibility ofreactive groups, by providing a mobile phase for reactants, and byserving as a reactant in several deleterious processes such asbeta-elimination and hydrolysis.

[0131] Protein preparations containing between about 6% to 28% water arethe most unstable. Below this level, the mobility of bound water andprotein internal motions are low. Above this level, water mobility andprotein motions approach those of full hydration. Up to a point,increased susceptibility toward solid-phase aggregation with increasinghydration has been observed in several systems. However, at higher watercontent, less aggregation is observed because of the dilution effect.

[0132] In accordance with these principles, an effective method forstabilizing peptides and proteins against solid-state aggregation formucosal delivery is to control the water content in a solid formulationand maintain the water activity in the formulation at optimal levels.This level depends on the nature of the protein, but in general,proteins maintained below their “monolayer” water coverage will exhibitsuperior solid-state stability.

[0133] A variety of additives, diluents, bases and delivery vehicles areprovided within the invention that effectively control water content toenhance protein stability. These reagents and carrier materialseffective as anti-aggregation agents in this sense include, for example,polymers of various functionalities, such as polyethylene glycol,dextran, diethylaminoethyl dextran, and carboxymethyl cellulose, whichsignificantly increase the stability and reduce the solid-phaseaggregation of peptides and proteins admixed therewith or linkedthereto. In some instances, the activity or physical stability ofproteins can also be enhanced by various additives to aqueous solutionsof the peptide or protein drugs. For example, additives, such as polyols(including sugars), amino acids, proteins such as collagen and gelatin,and various salts may be used.

[0134] Certain additives, in particular sugars and other polyols, alsoimpart significant physical stability to dry, e.g., lyophilizedproteins. These additives can also be used within the invention toprotect the proteins against aggregation not only during lyophilizationbut also during storage in the dry state. For example sucrose and Ficoll70 (a polymer with sucrose units) exhibit significant protection againstpeptide or protein aggregation during solid-phase incubation undervarious conditions. These additives may also enhance the stability ofsolid proteins embedded within polymer matrices.

[0135] Yet additional additives, for example sucrose, stabilize proteinsagainst solid-state aggregation in humid atmospheres at elevatedtemperatures, as may occur in certain sustained-release formulations ofthe invention. Proteins such as gelatin and collagen also serve asstabilizing or bulking agents to reduce denaturation and aggregation ofunstable proteins in this context. These additives can be incorporatedinto polymeric melt processes and compositions within the invention. Forexample, polypeptide microparticles can be prepared by simplylyophilizing or spray drying a solution containing various stabilizingadditives described above. Sustained release of unaggregated peptidesand proteins can thereby be obtained over an extended period of time.

[0136] Various additional preparative components and methods, as well asspecific formulation additives, are provided herein which yieldformulations for mucosal delivery of aggregation-prone peptides andproteins, wherein the peptide or protein is stabilized in asubstantially pure, unaggregated form using a solubilization agent. Arange of components and additives are contemplated for use within thesemethods and formulations. Exemplary of these solubilization agents arecyclodextrins (CDs), which selectively bind hydrophobic side chains ofpolypeptides. These CDs have been found to bind to hydrophobic patchesof proteins in a manner that significantly inhibits aggregation. Thisinhibition is selective with respect to both the CD and the proteininvolved. Such selective inhibition of protein aggregation providesadditional advantages within the intranasal delivery methods andcompositions of the invention. Additional agents for use in this contextinclude CD dimers, trimers and tetramers with varying geometriescontrolled by the linkers that specifically block aggregation ofpeptides and protein. Yet solubilization agents and methods forincorporation within the invention involve the use of peptides andpeptide mimetics to selectively block protein-protein interactions. Inone aspect, the specific binding of hydrophobic side chains reported forCD multimers is extended to proteins via the use of peptides and peptidemimetics that similarly block protein aggregation. A wide range ofsuitable methods and anti-aggregation agents are available forincorporation within the compositions and procedures of the invention.

[0137] Charge Modifying and pH Control Agents and Methods

[0138] To improve the transport characteristics of biologically activeagents (including Y2 receptor-binding peptide, other active peptides andproteins, and macromolecular and small molecule drugs) for enhanceddelivery across hydrophobic mucosal membrane barriers, the inventionalso provides techniques and reagents for charge modification ofselected biologically active agents or delivery-enhancing agentsdescribed herein. In this regard, the relative permeabilities ofmacromolecules is generally be related to their partition coefficients.The degree of ionization of molecules, which is dependent on the pKa ofthe molecule and the pH at the mucosal membrane surface, also affectspermeability of the molecules. Permeation and partitioning ofbiologically active agents, including Y2 receptor-binding peptide andanalogs of the invention, for mucosal delivery may be facilitated bycharge alteration or charge spreading of the active agent orpermeabilizing agent, which is achieved, for example, by alteration ofcharged functional groups, by modifying the pH of the delivery vehicleor solution in which the active agent is delivered, or by coordinateadministration of a charge- or pH-altering reagent with the activeagent.

[0139] Consistent with these general teachings, mucosal delivery ofcharged macromolecular species, including Y2 receptor-binding peptideand other biologically active peptides and proteins, within the methodsand compositions of the invention is substantially improved when theactive agent is delivered to the mucosal surface in a substantiallyun-ionized, or neutral, electrical charge state.

[0140] Certain Y2 receptor-binding peptide and other biologically activepeptide and protein components of mucosal formulations for use withinthe invention will be charge modified to yield an increase in thepositive charge density of the peptide or protein. These modificationsextend also to cationization of peptide and protein conjugates, carriersand other delivery forms disclosed herein. Cationization offers aconvenient means of altering the biodistribution and transportproperties of proteins and macromolecules within the invention.Cationization is undertaken in a manner that substantially preserves thebiological activity of the active agent and limits potentially adverseside effects, including tissue damage and toxicity.

[0141] Degradative Enzyme Inhibitory Agents and Methods

[0142] Another excipient that may be included in a trans-mucosalpreparation is a degradative enzyme inhibitor. Exemplary mucoadhesivepolymer-enzyme inhibitor complexes that are useful within the mucosaldelivery formulations and methods of the invention include, but are notlimited to: Carboxymethylcellulose-pepstatin (with anti-pepsinactivity); Poly(acrylic acid)-Bowman-Birk inhibitor (anti-chymotrypsin);Poly(acrylic acid)-chymostatin (anti-chymotrypsin); Poly(acrylicacid)-elastatinal (anti-elastase); Carboxymethylcellulose-elastatinal(anti-elastase); Polycarbophil—elastatinal (anti-elastase);Chitosan—antipain (anti-trypsin); Poly(acrylic acid)—bacitracin(anti-aminopeptidase N); Chitosan—EDTA (anti-aminopeptidase N,anti-carboxypeptidase A); Chitosan—EDTA—antipain (anti-trypsin,anti-chymotrypsin, anti-elastase). As described in further detail below,certain embodiments of the invention will optionally incorporate a novelchitosan derivative or chemically modified form of chitosan. One suchnovel derivative for use within the invention is denoted as aβ-[1→4]-2-guanidino-2-deoxy-D-glucose polymer (poly-GuD).

[0143] Any inhibitor that inhibits the activity of an enzyme to protectthe biologically active agent(s) may be usefully employed in thecompositions and methods of the invention. Useful enzyme inhibitors forthe protection of biologically active proteins and peptides include, forexample, soybean trypsin inhibitor, pancreatic trypsin inhibitor,chymotrypsin inhibitor and trypsin and chrymotrypsin inhibitor isolatedfrom potato (solanum tuberosum L.) tubers. A combination or mixtures ofinhibitors may be employed. Additional inhibitors of proteolytic enzymesfor use within the invention include ovomucoid-enzyme, gabaxatemesylate, alpha1-antitrypsin, aprotinin, amastatin, bestatin, puromycin,bacitracin, leupepsin, alpha2-macroglobulin, pepstatin and egg white orsoybean trypsin inhibitor. These and other inhibitors can be used aloneor in combination. The inhibitor(s) may be incorporated in or bound to acarrier, e.g., a hydrophilic polymer, coated on the surface of thedosage form which is to contact the nasal mucosa, or incorporated in thesuperficial phase of the surface, in combination with the biologicallyactive agent or in a separately administered (e.g., pre-administered)formulation.

[0144] The amount of the inhibitor, e.g., of a proteolytic enzymeinhibitor that is optionally incorporated in the compositions of theinvention will vary depending on (a) the properties of the specificinhibitor, (b) the number of functional groups present in the molecule(which may be reacted to introduce ethylenic unsaturation necessary forcopolymerization with hydrogel forming monomers), and (c) the number oflectin groups, such as glycosides, which are present in the inhibitormolecule. It may also depend on the specific therapeutic agent that isintended to be administered. Generally speaking, a useful amount of anenzyme inhibitor is from about 0.1 mg/ml to about 50 mg/ml, often fromabout 0.2 mg/ml to about 25 mg/ml, and more commonly from about 0.5mg/ml to 5 mg/ml of the of the formulation (i.e., a separate proteaseinhibitor formulation or combined formulation with the inhibitor andbiologically active agent).

[0145] In the case of trypsin inhibition, suitable inhibitors may beselected from, e.g., aprotinin, BBI, soybean trypsin inhibitor, chickenovomucoid, chicken ovoinhibitor, human pancreatic trypsin inhibitor,camostat mesilate, flavonoid inhibitors, antipain, leupeptin,p-aminobenzamidine, AEBSF, TLCK (tosyllysine chloromethylketone), APMSF,DFP, PMSF, and poly(acrylate) derivatives. In the case of chymotrypsininhibition, suitable inhibitors may be selected from, e.g., aprotinin,BBI, soybean trypsin inhibitor, chymostatin,benzyloxycarbonyl-Pro-Phe-CHO, FK-448, chicken ovoinhibitor, sugarbiphenylboronic acids complexes, DFP, PMSF, β-phenylpropionate, andpoly(acrylate) derivatives. In the case of elastase inhibition, suitableinhibitors may be selected from, e.g., elastatinal,methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone (MPCMK), BBI, soybeantrypsin inhibitor, chicken ovoinhibitor, DFP, and PMSF.

[0146] Additional enzyme inhibitors for use within the invention areselected from a wide range of non-protein inhibitors that vary in theirdegree of potency and toxicity. As described in further detail below,immobilization of these adjunct agents to matrices or other deliveryvehicles, or development of chemically modified analogues, may bereadily implemented to reduce or even eliminate toxic effects, when theyare encountered. Among this broad group of candidate enzyme inhibitorsfor use within the invention are organophosphorous inhibitors, such asdiisopropylfluorophosphate (DFP) and phenylmethylsulfonyl fluoride(PMSF), which are potent, irreversible inhibitors of serine proteases(e.g., trypsin and chymotrypsin). The additional inhibition ofacetylcholinesterase by these compounds makes them highly toxic inuncontrolled delivery settings. Another candidate inhibitor,4-(2-Aminoethyl)-benzenesulfonyl fluoride (AEBSF), has an inhibitoryactivity comparable to DFP and PMSF, but it is markedly less toxic.(4-Aminophenyl)-methanesulfonyl fluoride hydrochloride (APMSF) isanother potent inhibitor of trypsin, but is toxic in uncontrolledsettings. In contrast to these inhibitors,4-(4-isopropylpiperadinocarbonyl)phenyl 1,2,3,4,-tetrahydro-1-naphthoate methanesulphonate (FK-448) is a low toxicsubstance, representing a potent and specific inhibitor of chymotrypsin.Further representatives of this non-protein group of inhibitorcandidates, and also exhibiting low toxic risk, are camostat mesilate(N,N′-dimethyl carbamoylmethyl-p-(p′-guanidino-benzoyloxy)phenylacetatemethane-sulphonate).

[0147] Yet another type of enzyme inhibitory agent for use within themethods and compositions of the invention are amino acids and modifiedamino acids that interfere with enzymatic degradation of specifictherapeutic compounds. For use in this context, amino acids and modifiedamino acids are substantially non-toxic and can be produced at a lowcost. However, due to their low molecular size and good solubility, theyare readily diluted and absorbed in mucosal environments. Nevertheless,under proper conditions, amino acids can act as reversible, competitiveinhibitors of protease enzymes. Certain modified amino acids can displaya much stronger inhibitory activity. A desired modified amino acid inthis context is known as a ‘transition-state’ inhibitor. The stronginhibitory activity of these compounds is based on their structuralsimilarity to a substrate in its transition-state geometry, while theyare generally selected to have a much higher affinity for the activesite of an enzyme than the substrate itself. Transition-state inhibitorsare reversible, competitive inhibitors. Examples of this type ofinhibitor are α-aminoboronic acid derivatives, such as boro-leucine,boro-valine and boro-alanine. The boron atom in these derivatives canform a tetrahedral boronate ion that is believed to resemble thetransition state of peptides during their hydrolysis by aminopeptidases.These amino acid derivatives are potent and reversible inhibitors ofaminopeptidases and it is reported that boro-leucine is more than100-times more effective in enzyme inhibition than bestatin and morethan 1000-times more effective than puromycin. Another modified aminoacid for which a strong protease inhibitory activity has been reportedis N-acetylcysteine, which inhibits enzymatic activity of aminopeptidaseN. This adjunct agent also displays mucolytic properties that can beemployed within the methods and compositions of the invention to reducethe effects of the mucus diffusion barrier.

[0148] Still other useful enzyme inhibitors for use within thecoordinate administration methods and combinatorial formulations of theinvention may be selected from peptides and modified peptide enzymeinhibitors. An important representative of this class of inhibitors isthe cyclic dodecapeptide, bacitracin, obtained from Bacilluslicheniformis. In addition to these types of peptides, certaindipeptides and tripeptides display weak, non-specific inhibitoryactivity towards some protease. By analogy with amino acids, theirinhibitory activity can be improved by chemical modifications. Forexample, phosphinic acid dipeptide analogues are also ‘transition-state’inhibitors with a strong inhibitory activity towards aminopeptidases.They have reportedly been used to stabilize nasally administered leucineenkephalin. Another example of a transition-state analogue is themodified pentapeptide pepstatin, which is a very potent inhibitor ofpepsin. Structural analysis of pepstatin, by testing the inhibitoryactivity of several synthetic analogues, demonstrated the majorstructure-function characteristics of the molecule responsible for theinhibitory activity. Another special type of modified peptide includesinhibitors with a terminally located aldehyde function in theirstructure. For example, the sequence benzyloxycarbonyl-Pro-Phe-CHO,which fulfills the known primary and secondary specificity requirementsof chymotrypsin, has been found to be a potent reversible inhibitor ofthis target proteinase. The chemical structures of further inhibitorswith a terminally located aldehyde function, e.g. antipain, leupeptin,chymostatin and elastatinal, are also known in the art, as are thestructures of other known, reversible, modified peptide inhibitors, suchas phosphoramidon, bestatin, puromycin and amastatin.

[0149] Due to their comparably high molecular mass, polypeptide proteaseinhibitors are more amenable than smaller compounds to concentrateddelivery in a drug-carrier matrix. Additional agents for proteaseinhibition within the formulations and methods of the invention involvethe use of complexing agents. These agents mediate enzyme inhibition bydepriving the intranasal environment (or preparative or therapeuticcomposition) of divalent cations, which are co-factors for manyproteases. For instance, the complexing agents EDTA and DTPA ascoordinately administered or combinatorially formulated adjunct agents,in suitable concentration, will be sufficient to inhibit selectedproteases to thereby enhance intranasal delivery of biologically activeagents according to the invention. Further representatives of this classof inhibitory agents are EGTA, 1,10-phenanthroline and hydroxychinoline.In addition, due to their propensity to chelate divalent cations, theseand other complexing agents are useful within the invention as direct,absorption-promoting agents.

[0150] As noted in more detail elsewhere herein, it is also contemplatedto use various polymers, particularly mucoadhesive polymers, as enzymeinhibiting agents within the coordinate administration, multi-processingand/or combinatorial formulation methods and compositions of theinvention. For example, poly(acrylate) derivatives, such as poly(acrylicacid) and polycarbophil, can affect the activity of various proteases,including trypsin, chymotrypsin. The inhibitory effect of these polymersmay also be based on the complexation of divalent cations such as Ca²⁺and Zn²⁺. It is further contemplated that these polymers may serve asconjugate partners or carriers for additional enzyme inhibitory agents,as described above. For example, a chitosan-EDTA conjugate has beendeveloped and is useful within the invention that exhibits a stronginhibitory effect towards the enzymatic activity of zinc-dependentproteases. The mucoadhesive properties of polymers following covalentattachment of other enzyme inhibitors in this context are not expectedto be substantially compromised, nor is the general utility of suchpolymers as a delivery vehicle for biologically active agents within theinvention expected to be diminished. On the contrary, the reduceddistance between the delivery vehicle and mucosal surface afforded bythe mucoadhesive mechanism will minimize presystemic metabolism of theactive agent, while the covalently bound enzyme inhibitors remainconcentrated at the site of drug delivery, minimizing undesired dilutioneffects of inhibitors as well as toxic and other side effects causedthereby. In this manner, the effective amount of a coordinatelyadministered enzyme inhibitor can be reduced due to the exclusion ofdilution effects.

[0151] Exemplary mucoadhesive polymer-enzyme inhibitor complexes thatare useful within the mucosal formulations and methods of the inventioninclude, but are not limited to: Carboxymethylcellulose-pepstatin (withanti-pepsin activity); Poly(acrylic acid)-Bowman-Birk inhibitor(anti-chymotrypsin); Poly(acrylic acid)-chymostatin (anti-chymotrypsin);Poly(acrylic acid)-elastatinal (anti-elastase);Carboxymethylcellulose-elastatinal (anti-elastase);Polycarbophil—lastatinal (anti-elastase); Chitosan—antipain(anti-trypsin); Poly(acrylic acid)—bacitracin (anti-aminopeptidase N);Chitosan-EDTA (anti-aminopeptidase N, anti-carboxypeptidase A);Chitosan—EDTA-antipain (anti-trypsin, anti-chymotrypsin, anti-elastase).

[0152] Mucolytic and Mucus-Clearing Agents and Methods

[0153] Effective delivery of biotherapeutic agents via intranasaladministration must take into account the decreased drug transport rateacross the protective mucus lining of the nasal mucosa, in addition todrug loss due to binding to glycoproteins of the mucus layer. Normalmucus is a viscoelastic, gel-like substance consisting of water,electrolytes, mucins, macromolecules, and sloughed epithelial cells. Itserves primarily as a cytoprotective and lubricative covering for theunderlying mucosal tissues. Mucus is secreted by randomly distributedsecretory cells located in the nasal epithelium and in other mucosalepithelia. The structural unit of mucus is mucin. This glycoprotein ismainly responsible for the viscoelastic nature of mucus, although othermacromolecules may also contribute to this property. In airway mucus,such macromolecules include locally produced secretory IgA, IgM, IgE,lysozyme, and bronchotransferrin, which also play an important role inhost defense mechanisms.

[0154] The coordinate administration methods of the instant inventionoptionally incorporate effective mucolytic or mucus-clearing agents,which serve to degrade, thin or clear mucus from intranasal mucosalsurfaces to facilitate absorption of intranasally administeredbiotherapeutic agents. Within these methods, a mucolytic ormucus-clearing agent is coordinately administered as an adjunct compoundto enhance intranasal delivery of the biologically active agent.Alternatively, an effective amount of a mucolytic or mucus-clearingagent is incorporated as a processing agent within a multi-processingmethod of the invention, or as an additive within a combinatorialformulation of the invention, to provide an improved formulation thatenhances intranasal delivery of biotherapeutic compounds by reducing thebarrier effects of intranasal mucus.

[0155] A variety of mucolytic or mucus-clearing agents are available forincorporation within the methods and compositions of the invention.Based on their mechanisms of action, mucolytic and mucus clearing agentscan often be classified into the following groups: proteases (e.g.,pronase, papain) that cleave the protein core of mucin glycoproteins;sulfhydryl compounds that split mucoprotein disulfide linkages; anddetergents (e.g., Triton X-100, Tween 20) that break non-covalent bondswithin the mucus. Additional compounds in this context include, but arenot limited to, bile salts and surfactants, for example, sodiumdeoxycholate, sodium taurodeoxycholate, sodium glycocholate, andlysophosphatidylcholine.

[0156] The effectiveness of bile salts in causing structural breakdownof mucus is in the order deoxycholate>taurocholate>glycocholate. Othereffective agents that reduce mucus viscosity or adhesion to enhanceintranasal delivery according to the methods of the invention include,e.g., short-chain fatty acids, and mucolytic agents that work bychelation, such as N-acylcollagen peptides, bile acids, and saponins(the latter function in part by chelating Ca²⁺ and/or Mg²⁺ which play animportant role in maintaining mucus layer structure).

[0157] Additional mucolytic agents for use within the methods andcompositions of the invention include N-acetyl-L-cysteine (ACS), apotent mucolytic agent that reduces both the viscosity and adherence ofbronchopulmonary mucus and is reported to modestly increase nasalbioavailability of human growth hormone in anesthetized rats (from 7.5to 12.2%). These and other mucolytic or mucus-clearing agents arecontacted with the nasal mucosa, typically in a concentration range ofabout 0.2 to 20 mM, coordinately with administration of the biologicallyactive agent, to reduce the polar viscosity and/or elasticity ofintranasal mucus.

[0158] Still other mucolytic or mucus-clearing agents may be selectedfrom a range of glycosidase enzymes, which are able to cleave glycosidicbonds within the mucus glycoprotein. α-amylase and β-amylase arerepresentative of this class of enzymes, although their mucolytic effectmay be limited. In contrast, bacterial glycosidases which allow thesemicroorganisms to permeate mucus layers of their hosts.

[0159] For combinatorial use with most biologically active agents withinthe invention, including peptide and protein therapeutics, non-ionogenicdetergents are generally also useful as mucolytic or mucus-clearingagents. These agents typically will not modify or substantially impairthe activity of therapeutic polypeptides.

[0160] Ciliostatic Agents and Methods

[0161] Because the self-cleaning capacity of certain mucosal tissues(e.g., nasal mucosal tissues) by mucociliary clearance is necessary as aprotective function (e.g., to remove dust, allergens, and bacteria), ithas been generally considered that this function should not besubstantially impaired by mucosal medications. Mucociliary transport inthe respiratory tract is a particularly important defense mechanismagainst infections. To achieve this function, ciliary beating in thenasal and airway passages moves a layer of mucus along the mucosa toremoving inhaled particles and microorganisms.

[0162] Ciliostatic agents find use within the methods and compositionsof the invention to increase the residence time of mucosally (e.g.,intranasally) administered Y2 receptor-binding peptide, analogs andmimetics, and other biologically active agents disclosed herein. Inparticular, the delivery these agents within the methods andcompositions of the invention is significantly enhanced in certainaspects by the coordinate administration or combinatorial formulation ofone or more ciliostatic agents that function to reversibly inhibitciliary activity of mucosal cells, to provide for a temporary,reversible increase in the residence time of the mucosally administeredactive agent(s). For use within these aspects of the invention, theforegoing ciliostatic factors, either specific or indirect in theiractivity, are all candidates for successful employment as ciliostaticagents in appropriate amounts (depending on concentration, duration andmode of delivery) such that they yield a transient (i.e., reversible)reduction or cessation of mucociliary clearance at a mucosal site ofadministration to enhance delivery of Y2 receptor-binding peptide,analogs and mimetics, and other biologically active agents disclosedherein, without unacceptable adverse side effects.

[0163] Within more detailed aspects, a specific ciliostatic factor isemployed in a combined formulation or coordinate administration protocolwith one or more Y2 receptor-binding peptide proteins, analogs andmimetics, and/or other biologically active agents disclosed herein.Various bacterial ciliostatic factors isolated and characterized in theliterature may be employed within these embodiments of the invention.Ciliostatic factors from the bacterium Pseudomonas aeruginosa include aphenazine derivative, a pyo compound (2-alkyl-4-hydroxyquinolines), anda rhamnolipid (also known as a hemolysin). The pyo compound producedciliostasis at concentrations of 50 μg/ml and without obviousultrastructural lesions. The phenazine derivative also inhibited ciliarymotility but caused some membrane disruption, although at substantiallygreater concentrations of 400 μg/ml. Limited exposure of trachealexplants to the rhamnolipid resulted in ciliostasis, which wasassociated with altered ciliary membranes. More extensive exposure torhamnolipid was associated with removal of dynein arms from axonemes.

[0164] Surface Active Agents and Methods

[0165] Within more detailed aspects of the invention, one or moremembrane penetration-enhancing agents may be employed within a mucosaldelivery method or formulation of the invention to enhance mucosaldelivery of Y2 receptor-binding peptide proteins, analogs and mimetics,and other biologically active agents disclosed herein. Membranepenetration enhancing agents in this context can be selected from: (i) asurfactant, (ii) a bile salt, (iii) a phospholipid additive, mixedmicelle, liposome, or carrier, (iv) an alcohol, (v) an enamine, (vi) anNO donor compound, (vii) a long-chain amphipathic molecule (viii) asmall hydrophobic penetration enhancer; (ix) sodium or a salicylic acidderivative; (x) a glycerol ester of acetoacetic acid (xi) aclyclodextrin or beta-cyclodextrin derivative, (xii) a medium-chainfatty acid, (xiii) a chelating agent, (xiv) an amino acid or saltthereof, (xv) an N-acetylamino acid or salt thereof, (xvi) an enzymedegradative to a selected membrane component, (xvii) an inhibitor offatty acid synthesis, or (xviii) an inhibitor of cholesterol synthesis;or (xix) any combination of the membrane penetration enhancing agentsrecited in (i)-(xix).

[0166] Certain surface-active agents are readily incorporated within themucosal delivery formulations and methods of the invention as mucosalabsorption enhancing agents. These agents, which may be coordinatelyadministered or combinatorially formulated with Y2 receptor-bindingpeptide proteins, analogs and mimetics, and other biologically activeagents disclosed herein, may be selected from a broad assemblage ofknown surfactants. Surfactants, which generally fall into three classes:(1) nonionic polyoxyethylene ethers; (2) bile salts such as sodiumglycocholate (SGC) and deoxycholate (DOC); and (3) derivatives offusidic acid such as sodium taurodihydrofusidate (STDHF). The mechanismsof action of these various classes of surface-active agents typicallyinclude solubilization of the biologically active agent. For proteinsand peptides which often form aggregates, the surface active propertiesof these absorption promoters can allow interactions with proteins suchthat smaller units such as surfactant coated monomers may be morereadily maintained in solution. Examples of other surface-active agentsare L-α-Phospharidycholine Didecanoyl (DDPC) polysorbate 80 andpolysorbate 20. These monomers are presumably more transportable unitsthan aggregates. A second potential mechanism is the protection of thepeptide or protein from proteolyfic degradation by proteases in themucosal environment. Both bile salts and some fusidic acid derivativesreportedly inhibit proteolytic degradation of proteins by nasalhomogenates at concentrations less than or equivalent to those requiredto enhance protein absorption. This protease inhibition may beespecially important for peptides with short biological half-lives.

[0167] Degradation Enzymes and Inhibitors of Fatty Acid and CholesterolSynthesis

[0168] In related aspects of the invention, Y2 receptor-binding peptideproteins, analogs and mimetics, and other biologically active agents formucosal administration are formulated or coordinately administered witha penetration enhancing agent selected from a degradation enzyme, or ametabolic stimulatory agent or inhibitor of synthesis of fatty acids,sterols or other selected epithelial barrier components, U.S. Pat. No.6,190,894. For example, degradative enzymes such as phospholipase,hyaluronidase, neuraminidase, and chondroitinase may be employed toenhance mucosal penetration of Y2 receptor-binding peptide proteins,analogs and mimetics, and other biologically active agent withoutcausing irreversible damage to the mucosal barrier. In one embodiment,chondroitinase is employed within a method or composition as providedherein to alter glycoprotein or glycolipid constituents of thepermeability barrier of the mucosa, thereby enhancing mucosal absorptionof Y2 receptor-binding peptide proteins, analogs and mimetics, and otherbiologically active agents disclosed herein.

[0169] With regard to inhibitors of synthesis of mucosal barrierconstituents, it is noted that free fatty acids account for 20-25% ofepithelial lipids by weight. Two rate-limiting enzymes in thebiosynthesis of free fatty acids are acetyl CoA carboxylase and fattyacid synthetase. Through a series of steps, free fatty acids aremetabolized into phospholipids. Thus, inhibitors of free fatty acidsynthesis and metabolism for use within the methods and compositions ofthe invention include, but are not limited to, inhibitors of acetyl CoAcarboxylase such as 5-tetradecyloxy-2-furancarboxylic acid (TOFA);inhibitors of fatty acid synthetase; inhibitors of phospholipase A suchas gomisin A, 2-(p-amylcinnamyl)amino-4-chlorobenzoic acid,bromophenacyl bromide, monoalide, 7,7-dimethyl-5,8-eicosadienoic acid,nicergoline, cepharanthine, nicardipine, quercetin, dibutyryl-cyclicAMP, R-24571, N-oleoylethanolamine, N-(7-nitro-2,1,3-benzoxadiazol4-yl)phosphostidyl serine, cyclosporine A, topical anesthetics, includingdibucaine, prenylamine, retinoids, such as all-trans and 13-cis-retinoicacid, W-7, trifluoperazine, R-24571 (calmidazolium),1-hexadocyl-3-trifluoroethyl glycero-sn-2-phosphomenthol (MJ33); calciumchannel blockers including nicardipine, verapamil, diltiazem,nifedipine, and nimodipine; antimalarials including quinacrine,mepacrine, chloroquine and hydroxychloroquine; beta blockers includingpropanalol and labetalol; calmodulin antagonists; EGTA; thimersol;glucocorticosteroids including dexamethasone and prednisolone; andnonsteroidal antiinflammatory agents including indomethacin andnaproxen.

[0170] Free sterols, primarily cholesterol, account for 20-25% of theepithelial lipids by weight. The rate limiting enzyme in thebiosynthesis of cholesterol is 3-hydroxy-3-methylglutaryl (HMG) CoAreductase. Inhibitors of cholesterol synthesis for use within themethods and compositions of the invention include, but are not limitedto, competitive inhibitors of (HMG) CoA reductase, such as simvastatin,lovastatin, fluindostatin (fluvastatin), pravastatin, mevastatin, aswell as other HMG CoA reductase inhibitors, such as cholesterol oleate,cholesterol sulfate and phosphate, and oxygenated sterols, such as25-OH—and 26-OH—cholesterol; inhibitors of squalene synthetase;inhibitors of squalene epoxidase; inhibitors of DELTA7 or DELTA24reductases such as 22,25-diazacholesterol, 20,25-diazacholestenol,AY9944, and triparanol.

[0171] Each of the inhibitors of fatty acid synthesis or the sterolsynthesis inhibitors may be coordinately administered or combinatoriallyformulated with one or more Y2 receptor-binding peptide proteins,analogs and mimetics, and other biologically active agents disclosedherein to achieve enhanced epithelial penetration of the activeagent(s). An effective concentration range for the sterol inhibitor in atherapeutic or adjunct formulation for mucosal delivery is generallyfrom about 0.0001% to about 20% by weight of the total, more typicallyfrom about 0.01% to about 5%.

[0172] Nitric Oxide Donor Agents and Methods

[0173] Within other related aspects of the invention, a nitric oxide(NO) donor is selected as a membrane penetration-enhancing agent toenhance mucosal delivery of one or more Y2 receptor-binding peptideproteins, analogs and mimetics, and other biologically active agentsdisclosed herein. Various NO donors are known in the art and are usefulin effective concentrations within the methods and formulations of theinvention. Exemplary NO donors include, but are not limited to,nitroglycerine, nitropruside, NOC5[3-(2-hydroxy-1-(methyl-ethyl)-2-nitrosohydrazino)-1-propanamine], NOC12 [N-ethyl-2-(1-ethyl-hydroxy-2-nitrosohydrazino)-ethanamine], SNAP[S-nitroso-N-acetyl-DL-penicillamine], NOR1 and NOR4. Within the methodsand compositions of the invention, an effective amount of a selected NOdonor is coordinately administered or combinatorially formulated withone or more Y2 receptor-binding peptide proteins, analogs and mimetics,and/or other biologically active agents disclosed herein, into orthrough the mucosal epithelium.

[0174] Agents for Modulating Epithelial Junction Structure and/orPhysiology

[0175] The present invention provides pharmaceutical composition thatcontains one or more Y2 receptor-binding peptide proteins, analogs ormimetics, and/or other biologically active agents in combination withmucosal delivery enhancing agents disclosed herein formulated in apharmaceutical preparation for mucosal delivery.

[0176] The permeabilizing agent reversibly enhances mucosal epithelialparacellular transport, typically by modulating epithelial junctionalstructure and/or physiology at a mucosal epithelial surface in thesubject. This effect typically involves inhibition by the permeabilizingagent of homotypic or heterotypic binding between epithelial membraneadhesive proteins of neighboring epithelial cells. Target proteins forthis blockade of homotypic or heterotypic binding can be selected fromvarious related junctional adhesion molecules (JAMs), occludins, orclaudins. Examples of this are antibodies, antibody fragments orsingle-chain antibodies that bind to the extracellular domains of theseproteins.

[0177] In yet additional detailed embodiments, the invention providespermeabilizing peptides and peptide analogs and mimetics for enhancingmucosal epithelial paracellular transport. The subject peptides andpeptide analogs and mimetics typically work within the compositions andmethods of the invention by modulating epithelial junctional structureand/or physiology in a mammalian subject. In certain embodiments, thepeptides and peptide analogs and mimetics effectively inhibit homotypicand/or heterotypic binding of an epithelial membrane adhesive proteinselected from a junctional adhesion molecule (JAM), occludin, orclaudin.

[0178] One such agent that has been extensively studied is the bacterialtoxin from Vibrio cholerae known as the “zonula occludens toxin” (ZOT).This toxin mediates increased intestinal mucosal permeability and causesdisease symptoms including diarrhea in infected subjects. Fasano et al,Proc. Nat. Acad. Sci., U.S.A., 8:5242-5246 (1991). When tested on rabbitileal mucosa, ZOT increased the intestinal permeability by modulatingthe structure of intercellular tight junctions. More recently, it hasbeen found that ZOT is capable of reversibly opening tight junctions inthe intestinal mucosa. It has also been reported that ZOT is capable ofreversibly opening tight junctions in the nasal mucosa. U.S. Pat. No.5,908,825.

[0179] Within the methods and compositions of the invention, ZOT, aswell as various analogs and mimetics of ZOT that function as agonists orantagonists of ZOT activity, are useful for enhancing intranasaldelivery of biologically active agents—by increasing paracellularabsorption into and across the nasal mucosa. In this context, ZOTtypically acts by causing a structural reorganization of tight junctionsmarked by altered localization of the junctional protein ZO1. Withinthese aspects of the invention, ZOT is coordinately administered orcombinatorially formulated with the biologically active agent in aneffective amount to yield significantly enhanced absorption of theactive agent, by reversibly increasing nasal mucosal permeabilitywithout substantial adverse side effects

[0180] Vasodilator Agents and Methods

[0181] Yet another class of absorption-promoting agents that showsbeneficial utility within the coordinate administration andcombinatorial formulation methods and compositions of the invention arevasoactive compounds, more specifically vasodilators. These compoundsfunction within the invention to modulate the structure and physiologyof the submucosal vasculature, increasing the transport rate of Y2receptor-binding peptide, analogs and mimetics, and other biologicallyactive agents into or through the mucosal epithelium and/or to specifictarget tissues or compartments (e.g., the systemic circulation orcentral nervous system.).

[0182] Vasodilator agents for use within the invention typically causesubmucosal blood vessel relaxation by either a decrease in cytoplasmiccalcium, an increase in nitric oxide (NO) or by inhibiting myosin lightchain kinase. They are generally divided into 9 classes: calciumantagonists, potassium channel openers, ACE inhibitors, angiotensin-IIreceptor antagonists, α-adrenergic and imidazole receptor antagonists,β1-adrenergic agonists, phosphodiesterase inhibitors, eicosanoids and NOdonors.

[0183] Despite chemical differences, the pharmacokinetic properties ofcalcium antagonists are similar. Absorption into the systemiccirculation is high, and these agents therefore undergo considerablefirst-pass metabolism by the liver, resulting in individual variation inpharmacokinetics. Except for the newer drugs of the dihydropyridine type(amlodipine, felodipine, isradipine, nilvadipine, nisoldipine andnitrendipine), the half-life of calcium antagonists is short. Therefore,to maintain an effective drug concentration for many of these mayrequire delivery by multiple dosing, or controlled release formulations,as described elsewhere herein. Treatment with the potassium channelopener minoxidil may also be limited in manner and level ofadministration due to potential adverse side effects.

[0184] ACE inhibitors prevent conversion of angiotensin-I toangiotensin-II, and are most effective when renin production isincreased. Since ACE is identical to kininase-II, which inactivates thepotent endogenous vasodilator bradykinin, ACE inhibition causes areduction in bradykinin degradation. ACE inhibitors provide the addedadvantage of cardioprotective and cardioreparative effects, bypreventing and reversing cardiac fibrosis and ventricular hypertrophy inanimal models. The predominant elimination pathway of most ACEinhibitors is via renal excretion. Therefore, renal impairment isassociated with reduced elimination and a dosage reduction of 25 to 50%is recommended in patients with moderate to severe renal impairment.

[0185] With regard to NO donors, these compounds are particularly usefulwithin the invention for their additional effects on mucosalpermeability. In addition to the above-noted NO donors, complexes of NOwith nucleophiles called NO/nucleophiles, or NONOates, spontaneously andnonenzymatically release NO when dissolved in aqueous solution atphysiologic pH. In contrast, nitro vasodilators such as nitroglycerinrequire specific enzyme activity for NO release. NONOates release NOwith a defined stoichiometry and at predictable rates ranging from <3minutes for diethylamine/NO to approximately 20 hours fordiethylenetriamine/NO (DETANO).

[0186] Within certain methods and compositions of the invention, aselected vasodilator agent is coordinately administered (e.g.,systemically or intranasally, simultaneously or in combinatoriallyeffective temporal association) or combinatorially formulated with oneor more Y2 receptor-binding peptide, analogs and mimetics, and otherbiologically active agent(s) in an amount effective to enhance themucosal absorption of the active agent(s) to reach a target tissue orcompartment in the subject (e.g., the liver, hepatic portal vein, CNStissue or fluid, or blood plasma).

[0187] Selective Transport-Enhancing Agents and Methods

[0188] The compositions and delivery methods of the invention optionallyincorporate a selective transport-enhancing agent that facilitatestransport of one or more biologically active agents. Thesetransport-enhancing agents may be employed in a combinatorialformulation or coordinate administration protocol with one or more ofthe Y2 receptor-binding peptide proteins, analogs and mimetics disclosedherein, to coordinately enhance delivery of one or more additionalbiologically active agent(s) across mucosal transport barriers, toenhance mucosal delivery of the active agent(s) to reach a target tissueor compartment in the subject (e.g., the mucosal epithelium, liver, CNStissue or fluid, or blood plasma). Alternatively, thetransport-enhancing agents may be employed in a combinatorialformulation or coordinate administration protocol to directly enhancemucosal delivery of one or more of the Y2 receptor-binding peptideproteins, analogs and mimetics, with or without enhanced delivery of anadditional biologically active agent.

[0189] Exemplary selective transport-enhancing agents for use withinthis aspect of the invention include, but are not limited to,glycosides, sugar-containing molecules, and binding agents such aslectin binding agents, which are known to interact specifically withepithelial transport barrier components. For example, specific“bioadhesive” ligands, including various plant and bacterial lectins,which bind to cell surface sugar moieties by receptor-mediatedinteractions can be employed as carriers or conjugated transportmediators for enhancing mucosal, e.g., nasal delivery of biologicallyactive agents within the invention. Certain bioadhesive ligands for usewithin the invention will mediate transmission of biological signals toepithelial target cells that trigger selective uptake of the adhesiveligand by specialized cellular transport processes (endocytosis ortranscytosis). These transport mediators can therefore be employed as a“carrier system” to stimulate or direct selective uptake of one or moreY2 receptor-binding peptide proteins, analogs and mimetics, and otherbiologically active agent(s) into and/or through mucosal epithelia.These and other selective transport-enhancing agents significantlyenhance mucosal delivery of macromolecular biopharmaceuticals(particularly peptides, proteins, oligonucleotides and polynucleotidevectors) within the invention. Lectins are plant proteins that bind tospecific sugars found on the surface of glycoproteins and glycolipids ofeukaryotic cells. Concentrated solutions of lectins have a‘mucotractive’ effect, and various studies have demonstrated rapidreceptor mediated endocytocis (RME) of lectins and lectin conjugates(e.g., concanavalin A conjugated with colloidal gold particles) acrossmucosal surfaces. Additional studies have reported that the uptakemechanisms for lectins can be utilized for intestinal drug targeting invivo. In certain of these studies, polystyrene nanoparticles (500 nm)were covalently coupled to tomato lectin and reported yielded improvedsystemic uptake after oral administration to rats.

[0190] In addition to plant lectins, microbial adhesion and invasionfactors provide a rich source of candidates for use asadhesive/selective transport carriers within the mucosal deliverymethods and compositions of the invention. Two components are necessaryfor bacterial adherence processes, a bacterial ‘adhesin’ (adherence orcolonization factor) and a receptor on the host cell surface. Bacteriacausing mucosal infections need to penetrate the mucus layer beforeattaching themselves to the epithelial surface. This attachment isusually mediated by bacterial fimbriae or pilus structures, althoughother cell surface components may also take part in the process.Adherent bacteria colonize mucosal epithelia by multiplication andinitiation of a series of biochemical reactions inside the target cellthrough signal transduction mechanisms (with or without the help oftoxins). Associated with these invasive mechanisms, a wide diversity ofbioadhesive proteins (e.g., invasin, internalin) originally produced byvarious bacteria and viruses are known. These allow for extracellularattachment of such microorganisms with an impressive selectivity forhost species and even particular target tissues. Signals transmitted bysuch receptor-ligand interactions trigger the transport of intact,living microorganisms into, and eventually through, epithelial cells byendo- and transcytotic processes. Such naturally occurring phenomena maybe harnessed (e.g., by complexing biologically active agents such as Y2receptor-binding peptide with an adhesin) according to the teachingsherein for enhanced delivery of biologically active compounds into oracross mucosal epithelia and/or to other designated target sites of drugaction.

[0191] Various bacterial and plant toxins that bind epithelial surfacesin a specific, lectin-like manner are also useful within the methods andcompositions of the invention. For example, diptheria toxin (DT) entershost cells rapidly by RME. Likewise, the B subunit of the E. coli heatlabile toxin binds to the brush border of intestinal epithelial cells ina highly specific, lectin-like manner. Uptake of this toxin andtranscytosis to the basolateral side of the enterocytes has beenreported in vivo and in vitro. Other researches have expressed thetransmembrane domain of diphtheria toxin in E. coli as a maltose-bindingfusion protein and coupled it chemically to high-Mw poly-L-lysine. Theresulting complex was successfully used to mediate internalization of areporter gene in vitro. In addition to these examples, Staphylococcusaureus produces a set of proteins (e.g., staphylococcal enterotoxin A(SEA), SEB, toxic shock syndrome toxin I (TSST-1) which act both assuperantigens and toxins. Studies relating to these proteins havereported dose-dependent, facilitated transcytosis of SEB and TSST-I inCaco-2 cells.

[0192] Viral haemagglutinins comprise another type of transport agent tofacilitate mucosal delivery of biologically active agents within themethods and compositions of the invention. The initial step in manyviral infections is the binding of surface proteins (haemagglutinins) tomucosal cells. These binding proteins have been identified for mostviruses, including rotaviruses, varicella zoster virus, semliki forestvirus, adenoviruses, potato leafroll virus, and reovirus. These andother exemplary viral hemagglutinins can be employed in a combinatorialformulation (e.g., a mixture or conjugate formulation) or coordinateadministration protocol with one or more of the Y2 receptor-bindingpeptide, analogs and mimetics disclosed herein, to coordinately enhancemucosal delivery of one or more additional biologically active agent(s).Alternatively, viral hemagglutinins can be employed in a combinatorialformulation or coordinate administration protocol to directly enhancemucosal delivery of one or more of the Y2 receptor-binding peptideproteins, analogs and mimetics, with or without enhanced delivery of anadditional biologically active agent.

[0193] A variety of endogenous, selective transport-mediating factorsare also available for use within the invention. Mammalian cells havedeveloped an assortment of mechanisms to facilitate the internalizationof specific substrates and target these to defined compartments.Collectively, these processes of membrane deformations are termed‘endocytosis’ and comprise phagocytosis, pinocytosis, receptor-mediatedendocytosis (clathrin-mediated RME), and potocytosis(non-clathrin-mediated RME). RME is a highly specific cellular biologicprocess by which, as its name implies, various ligands bind to cellsurface receptors and are subsequently internalized and traffickedwithin the cell. In many cells the process of endocytosis is so activethat the entire membrane surface is internalized and replaced in lessthan a half hour. Two classes of receptors are proposed based on theirorientation in the cell membrane; the amino terminus of Type I receptorsis located on the extracellular side of the membrane, whereas Type IIreceptors have this same protein tail in the intracellular milieu.

[0194] Still other embodiments of the invention utilize transferrin as acarrier or stimulant of RME of mucosally delivered biologically activeagents. Transferrin, an 80 kDa iron-transporting glycoprotein, isefficiently taken up into cells by RME. Transferrin receptors are foundon the surface of most proliferating cells, in elevated numbers onerythroblasts and on many kinds of tumors. The transcytosis oftransferrin (Tf) and transferrin conjugates is reportedly enhanced inthe presence of Brefeldin A (BFA), a fungal metabolite. In otherstudies, BFA treatment has been reported to rapidly increase apicalendocytosis of both ricin and HRP in MDCK cells. Thus, BFA and otheragents that stimulate receptor-mediated transport can be employed withinthe methods of the invention as combinatorially formulated (e.g.,conjugated) and/or coordinately administered agents to enhancereceptor-mediated transport of biologically active agents, including Y2receptor-binding peptide proteins, analogs and mimetics.

[0195] Polymeric Delivery Vehicles and Methods

[0196] Within certain aspects of the invention, Y2 receptor-bindingpeptide proteins, analogs and mimetics, other biologically active agentsdisclosed herein, and delivery-enhancing agents as described above, are,individually or combinatorially, incorporated within a mucosally (e.g.,nasally) administered formulation that includes a biocompatible polymerfunctioning as a carrier or base. Such polymer carriers includepolymeric powders, matrices or microparticulate delivery vehicles, amongother polymer forms. The polymer can be of plant, animal, or syntheticorigin. Often the polymer is crosslinked. Additionally, in thesedelivery systems the Y2 receptor-binding peptide, analog or mimetic, canbe functionalized in a manner where it can be covalently bound to thepolymer and rendered inseparable from the polymer by simple washing. Inother embodiments, the polymer is chemically modified with an inhibitorof enzymes or other agents which may degrade or inactivate thebiologically active agent(s) and/or delivery enhancing agent(s). Incertain formulations, the polymer is a partially or completely waterinsoluble but water swellable polymer, e.g., a hydrogel. Polymers usefulin this aspect of the invention are desirably water interactive and/orhydrophilic in nature to absorb significant quantities of water, andthey often form hydrogels when placed in contact with water or aqueousmedia for a period of time sufficient to reach equilibrium with water.In more detailed embodiments, the polymer is a hydrogel which, whenplaced in contact with excess water, absorbs at least two times itsweight of water at equilibrium when exposed to water at roomtemperature, U.S. Pat. No. 6,004,583.

[0197] Drug delivery systems based on biodegradable polymers arepreferred in many biomedical applications because such systems arebroken down either by hydrolysis or by enzymatic reaction into non-toxicmolecules. The rate of degradation is controlled by manipulating thecomposition of the biodegradable polymer matrix. These types of systemscan therefore be employed in certain settings for long-term release ofbiologically active agents. Biodegradable polymers such as poly(glycolicacid) (PGA), poly-(lactic acid) (PLA), and poly(D,L-lactic-co-glycolicacid) (PLGA), have received considerable attention as possible drugdelivery carriers, since the degradation products of these polymers havebeen found to have low toxicity. During the normal metabolic function ofthe body these polymers degrade into carbon dioxide and water. Thesepolymers have also exhibited excellent biocompatibility.

[0198] For prolonging the biological activity of Y2 receptor-bindingpeptide, analogs and mimetics, and other biologically active agentsdisclosed herein, as well as optional delivery-enhancing agents, theseagents may be incorporated into polymeric matrices, e.g.,polyorthoesters, polyanhydrides, or polyesters. This yields sustainedactivity and release of the active agent(s), e.g., as determined by thedegradation of the polymer matrix. Although the encapsulation ofbiotherapeutic molecules inside synthetic polymers may stabilize themduring storage and delivery, the largest obstacle of polymer-basedrelease technology is the activity loss of the therapeutic moleculesduring the formulation processes that often involve heat, sonication ororganic solvents.

[0199] Absorption-promoting polymers contemplated for use within theinvention may include derivatives and chemically or physically modifiedversions of the foregoing types of polymers, in addition to othernaturally occurring or synthetic polymers, gums, resins, and otheragents, as well as blends of these materials with each other or otherpolymers, so long as the alterations, modifications or blending do notadversely affect the desired properties, such as water absorption,hydrogel formation, and/or chemical stability for useful application. Inmore detailed aspects of the invention, polymers such as nylon, acrylanand other normally hydrophobic synthetic polymers may be sufficientlymodified by reaction to become water swellable and/or form stable gelsin aqueous media.

[0200] Absorption-promoting polymers of the invention may includepolymers from the group of homo- and copolymers based on variouscombinations of the following vinyl monomers: acrylic and methacrylicacids, acrylamide, methacrylamide, hydroxyethylacrylate or methacrylate,vinylpyrrolidones, as well as polyvinylalcohol and its co- andterpolymers, polyvinylacetate, its co- and terpolymers with the abovelisted monomers and 2-acrylamido-2-methyl-propanesulfonic acid (AMPS®).Very useful are copolymers of the above listed monomers withcopolymerizable functional monomers such as acryl or methacryl amideacrylate or methacrylate esters where the ester groups are derived fromstraight or branched chain alkyl, aryl having up to four aromatic ringswhich may contain alkyl substituents of 1 to 6 carbons; steroidal,sulfates, phosphates or cationic monomers such asN,N-dimethylaminoalkyl(meth)acrylamide,dimethylaminoalkyl(meth)acrylate, (meth)acryloxyalkyltrimethylammoniumchloride, (meth)acryloxyalkyldimethylbenzyl ammonium chloride.

[0201] Additional absorption-promoting polymers for use within theinvention are those classified as dextrans, dextrins, and from the classof materials classified as natural gums and resins, or from the class ofnatural polymers such as processed collagen, chitin, chitosan, pullalan,zooglan, alginates and modified alginates such as “Kelcoloid” (apolypropylene glycol modified alginate) gellan gums such as “Kelocogel”,Xanathan gums such as “Keltrol”, estastin, alpha hydroxy butyrate andits copolymers, hyaluronic acid and its derivatives, polylactic andglycolic acids.

[0202] A very useful class of polymers applicable within the instantinvention are olefinically-unsaturated carboxylic acids containing atleast one activated carbon-to-carbon olefinic double bond, and at leastone carboxyl group; that is, an acid or functional group readilyconverted to an acid containing an olefinic double bond which readilyfunctions in polymerization because of its presence in the monomermolecule, either in the alpha-beta position with respect to a carboxylgroup, or as part of a terminal methylene grouping.Olefinically-unsaturated acids of this class include such materials asthe acrylic acids typified by the acrylic acid itself, alpha-cyanoacrylic acid, beta methylacrylic acid (crotonic acid), alpha-phenylacrylic acid, beta-acryloxy propionic acid, cinnamic acid, p-chlorocinnamic acid, 1-carboxy-4-phenyl butadiene-1,3, itaconic acid,citraconic acid, mesaconic acid, glutaconic acid, aconitic acid, maleicacid, fumaric acid, and tricarboxy ethylene. As used herein, the term“carboxylic acid” includes the polycarboxylic acids and those acidanhydrides, such as maleic anhydride, wherein the anhydride group isformed by the elimination of one molecule of water from two carboxylgroups located on the same carboxylic acid molecule.

[0203] Representative acrylates useful as absorption-promoting agentswithin the invention include methyl acrylate, ethyl acrylate, propylacrylate, isopropyl acrylate, butyl acrylate, isobutyl acrylate, methylmethacrylate, methyl ethacrylate, ethyl methacrylate, octyl acrylate,heptyl acrylate, octyl methacrylate, isopropyl methacrylate,2-ethylhexyl methacrylate, nonyl acrylate, hexyl acrylate, n-hexylmethacrylate, and the like. Higher alkyl acrylic esters are decylacrylate, isodecyl methacrylate, lauryl acrylate, stearyl acrylate,behenyl acrylate and melissyl acrylate and methacrylate versionsthereof. Mixtures of two or three or more long chain acrylic esters maybe successfully polymerized with one of the carboxylic monomers. Othercomonomers include olefins, including alpha olefins, vinyl ethers, vinylesters, and mixtures thereof.

[0204] Other vinylidene monomers, including the acrylic nitriles, mayalso be used as absorption-promoting agents within the methods andcompositions of the invention to enhance delivery and absorption of oneor more Y2 receptor-binding peptide proteins, analogs and mimetics, andother biologically active agent(s), including to enhance delivery of theactive agent(s) to a target tissue or compartment in the subject (e.g.,the liver, hepatic portal vein, CNS tissue or fluid, or blood plasma).Useful alpha, beta-olefinically unsaturated nitriles are preferablymonoolefinically unsaturated nitriles having from 3 to 10 carbon atomssuch as acrylonitrile, methacrylonitrile, and the like. Most preferredare acrylonitrile and methacrylonitrile. Acrylic amides containing from3 to 35 carbon atoms including monoolefinically unsaturated amides alsomay be used. Representative amides include acrylamide, methacrylamide,N-t-butyl acrylamide, N-cyclohexyl acrylamide, higher alkyl amides,where the alkyl group on the nitrogen contains from 8 to 32 carbonatoms, acrylic amides including N-alkylol amides of alpha,beta-olefinically unsaturated carboxylic acids including those havingfrom 4 to 10 carbon atoms such as N-methylol acrylamide, N-propanolacrylamide, N-methylol methacrylamide, N-methylol maleimide, N-methylolmaleamic acid esters, N-methylol-p-vinyl benzamide, and the like.

[0205] Yet additional useful absorption promoting materials arealpha-olefins containing from 2 to 18 carbon atoms, more preferably from2 to 8 carbon atoms; dienes containing from 4 to 10 carbon atoms; vinylesters and allyl esters such as vinyl acetate; vinyl aromatics such asstyrene, methyl styrene and chloro-styrene; vinyl and allyl ethers andketones such as vinyl methyl ether and methyl vinyl ketone;chloroacrylates; cyanoalkyl acrylates such as alpha-cyanomethylacrylate, and the alpha-, beta-, and gamma-cyanopropyl acrylates;alkoxyacrylates such as methoxy ethyl acrylate; haloacrylates aschloroethyl acrylate; vinyl halides and vinyl chloride, vinylidenechloride and the like; divinyls, diacrylates and other polyfunctionalmonomers such as divinyl ether, diethylene glycol diacrylate, ethyleneglycol dimethacrylate, methylene-bis-acrylamide, allylpentaerythritol,and the like; and bis (beta-haloalkyl) alkenyl phosphonates such asbis(beta-chloroethyl) vinyl phosphonate and the like as are known tothose skilled in the art. Copolymers wherein the carboxy containingmonomer is aminor constituent, and the other vinylidene monomers presentas major components are readily prepared in accordance with the methodsdisclosed herein.

[0206] When hydrogels are employed as absorption promoting agents withinthe invention, these may be composed of synthetic copolymers from thegroup of acrylic and methacrylic acids, acrylamide, methacrylamide,hydroxyethylacrylate (HEA) or methacrylate (HEMA), and vinylpyrrolidoneswhich are water interactive and swellable. Specific illustrativeexamples of useful polymers, especially for the delivery of peptides orproteins, are the following types of polymers: (meth)acrylamide and 0.1to 99 wt. % (meth)acrylic acid; (meth)acrylamides and 0.1-75 wt %(meth)acryloxyethyl trimethyammonium chloride; (meth)acrylamide and0.1-75 wt % (meth)acrylamide; acrylic acid and 0.1-75 wt %alkyl(meth)acrylates; (meth)acrylamide and 0.1-75 wt % AMPS.RTM.(trademark of Lubrizol Corp.); (meth)acrylamide and 0 to 30 wt %alkyl(meth)acrylamides and 0.1-75 wt % AMPS.RTM.; (meth)acrylamide and0.1-99 wt. % HEMA; (metb)acrylamide and 0.1 to 75 wt % HEMA and 0.1 to99%(meth)acrylic acid; (meth)acrylic acid and 0.1-99 wt % HEMA; 50 mole% vinyl ether and 50 mole % maleic anhydride; (meth)acrylamide and 0.1to 75 wt % (meth)acryloxyalky dimethyl benzylammonium chloride;(meth)acrylamide and 0.1 to 99 wt % vinyl pyrrolidone; (meth)acrylamideand 50 wt % vinyl pyrrolidone and 0.1-99.9 wt % (meth)acrylic acid;(meth)acrylic acid and 0.1 to 75 wt % AMPS.RTM. and 0.1-75 wt %alkyl(meth)acrylamide. In the above examples, alkyl means C₁ to C₃₀,preferably C₁ to C₂₂, linear and branched and C₄ to C₁₆ cyclic; where(meth) is used, it means that the monomers with and without the methylgroup are included. Other very useful hydrogel polymers are swellable,but insoluble versions of poly(vinyl pyrrolidone) starch, carboxymethylcellulose and polyvinyl alcohol.

[0207] Additional polymeric hydrogel materials useful within theinvention include (poly) hydroxyalkyl (meth)acrylate: anionic andcationic hydrogels: poly(electrolyte) complexes; poly(vinyl alcohols)having a low acetate residual: a swellable mixture of crosslinked agarand crosslinked carboxymethyl cellulose: a swellable compositioncomprising methyl cellulose mixed with a sparingly crosslinked agar; awater swellable copolymer produced by a dispersion of finely dividedcopolymer of maleic anhydride with styrene, ethylene, propylene, orisobutylene; a water swellable polymer of N-vinyl lactams; swellablesodium salts of carboxymethyl cellulose; and the like.

[0208] Other gelable, fluid imbibing and retaining polymers useful forforming the hydrophilic hydrogel for mucosal delivery of biologicallyactive agents within the invention include pectin; polysaccharides suchas agar, acacia, karaya, tragacenth, algins and guar and theircrosslinked versions; acrylic acid polymers, copolymers and saltderivatives, polyacrylamides; water swellable indene maleic anhydridepolymers; starch graft copolymers; acrylate type polymers and copolymerswith water absorbability of about 2 to 400 times its original weight;diesters of polyglucan; a mixture of crosslinked poly(vinyl alcohol) andpoly(N-vinyl-2-pyrrolidone); polyoxybutylene-polyethylene blockcopolymer gels; carob gum; polyester gels; poly urea gels; polyethergels; polyamide gels; polyimide gels; polypeptide gels; polyamino acidgels; poly cellulosic gels; crosslinked indene-maleic anhydride acrylatepolymers; and polysaccharides.

[0209] Synthetic hydrogel polymers for use within the invention may bemade by an infinite combination of several monomers in several ratios.The hydrogel can be crosslinked and generally possesses the ability toimbibe and absorb fluid and swell or expand to an enlarged equilibriumstate. The hydrogel typically swells or expands upon delivery to thenasal mucosal surface, absorbing about 2-5,5-10, 10-50, up to 50-100 ormore times fold its weight of water. The optimum degree of swellabilityfor a given hydrogel will be determined for different biologicallyactive agents depending upon such factors as molecular weight, size,solubility and diffusion characteristics of the active agent carried byor entrapped or encapsulated within the polymer, and the specificspacing and cooperative chain motion associated with each individualpolymer.

[0210] Hydrophilic polymers useful within the invention are waterinsoluble but water swellable. Such water-swollen polymers as typicallyreferred to as hydrogels or gels. Such gels may be conveniently producedfrom water-soluble polymer by the process of crosslinking the polymersby a suitable crosslinking agent. However, stable hydrogels may also beformed from specific polymers under defined conditions of pH,temperature and/or ionic concentration, according to know methods in theart. Typically the polymers are cross-linked, that is, cross-linked tothe extent that the polymers possess good hydrophilic properties, haveimproved physical integrity (as compared to non cross-linked polymers ofthe same or similar type) and exhibit improved ability to retain withinthe gel network both the biologically active agent of interest andadditional compounds for coadministration therewith such as a cytokineor enzyme inhibitor, while retaining the ability to release the activeagent(s) at the appropriate location and time.

[0211] Generally hydrogel polymers for use within the invention arecrosslinked with a difunctional cross-linking in the amount of from 0.01to 25 weight percent, based on the weight of the monomers forming thecopolymer, and more preferably from 0.1 to 20 weight percent and moreoften from 0.1 to 15 weight percent of the crosslinking agent. Anotheruseful amount of a crosslinking agent is 0.1 to 10 weight percent. Tri,tetra or higher multifunctional crosslinking agents may also beemployed. When such reagents are utilized, lower amounts may be requiredto attain equivalent crosslinking density, i.e., the degree ofcrosslinking, or network properties that are sufficient to containeffectively the biologically active agent(s).

[0212] The crosslinks can be covalent, ionic or hydrogen bonds with thepolymer possessing the ability to swell in the presence of watercontaining fluids. Such crosslinkers and crosslinking reactions areknown to those skilled in the art and in many cases are dependent uponthe polymer system. Thus a crosslinked network may be formed by freeradical copolymerization of unsaturated monomers. Polymeric hydrogelsmay also be formed by crosslinking preformed polymers by reactingfunctional groups found on the polymers such as alcohols, acids, amineswith such groups as glyoxal, formaldehyde or glutaraldehyde, bisanhydrides and the like.

[0213] The polymers also may be cross-linked with any polyene, e.g.decadiene or trivinyl cyclohexane; acrylamides, such asN,N-methylene-bis (acrylamide); polyfunctional acrylates, such astrimethylol propane triacrylate; or polyfunctional vinylidene monomercontaining at least 2 terminal CH₂<groups, including, for example,divinyl benzene, divinyl naphthlene, allyl acrylates and the like. Incertain embodiments, cross-linking monomers for use in preparing thecopolymers are polyalkenyl polyethers having more than one alkenyl ethergrouping per molecule, which may optionally possess alkenyl groups inwhich an olefinic double bond is present attached to a terminalmethylene grouping (e.g., made by the etherification of a polyhydricalcohol containing at least 2 carbon atoms and at least 2 hydroxylgroups). Compounds of this class may be produced by reacting an alkenylhalide, such as allyl chloride or allyl bromide, with a stronglyalkaline aqueous solution of one or more polyhydric alcohols. Theproduct may be a complex mixture of polyethers with varying numbers ofether groups. Efficiency of the polyether cross-linking agent increaseswith the number of potentially polymerizable groups on the molecule.Typically, polyethers containing an average of two or more alkenyl ethergroupings per molecule are used. Other cross-linking monomers includefor example, diallyl esters, dimethallyl ethers, allyl or methallylacrylates and acrylamides, tetravinyl silane, polyalkenyl methanes,diacrylates, and dimethacrylates, divinyl compounds such as divinylbenzene, polyallyl phosphate, diallyloxy compounds and phosphite estersand the like. Typical agents are allyl pentaerythritol, allyl sucrose,trimethylolpropane triacrylate, 1,6-hexanediol diacrylate,trimethylolpropane diallyl ether, pentaerythritol triacrylate,tetramethylene dimethacrylate, ethylene diacrylate, ethylenedimethacrylate, triethylene glycol dimethacrylate, and the like. Allylpentaerythritol, trimethylolpropane diallylether and allyl sucroseprovide suitable polymers. When the cross-linking agent is present, thepolymeric mixtures usually contain between about 0.01 to 20 weightpercent, e.g., 1%, 5%, or 10% or more by weight of cross-linking monomerbased on the total of carboxylic acid monomer, plus other monomers.

[0214] In more detailed aspects of the invention, mucosal delivery of Y2receptor-binding peptide, analogs and mimetics, and other biologicallyactive agents disclosed herein, is enhanced by retaining the activeagent(s) in a slow-release or enzymatically or physiologicallyprotective carrier or vehicle, for example a hydrogel that shields theactive agent from the action of the degradative enzymes. In certainembodiments, the active agent is bound by chemical means to the carrieror vehicle, to which may also be admixed or bound additional agents suchas enzyme inhibitors, cytokines, etc. The active agent may alternatelybe immobilized through sufficient physical entrapment within the carrieror vehicle, e.g., a polymer matrix.

[0215] Polymers such as hydrogels useful within the invention mayincorporate functional linked agents such as glycosides chemicallyincorporated into the polymer for enhancing intranasal bioavailabilityof active agents formulated therewith. Examples of such glycosides areglucosides, fructosides, galactosides, arabinosides, mannosides andtheir alkyl substituted derivatives and natural glycosides such asarbutin, phlorizin, amygdalin, digitonin, saponin, and indican. Thereare several ways in which a typical glycoside may be bound to a polymer.For example, the hydrogen of the hydroxyl groups of a glycoside or othersimilar carbohydrate may be replaced by the alkyl group from a hydrogelpolymer to form an ether. Also, the hydroxyl groups of the glycosidesmay be reacted to esterify the carboxyl groups of a polymeric hydrogelto form polymeric esters in situ. Another approach is to employcondensation of acetobromoglucose with cholest-5-en-3beta-ol on acopolymer of maleic acid. N-substituted polyacrylamides can besynthesized by the reaction of activated polymers withomega-aminoalkylglycosides: (1) (carbohydrate-spacer)(n)-polyacrylamide,‘pseudopolysaccharides’; (2) (carbohydratespacer)(n)-phosphatidylethanolamine(m)-polyacrylamide, neoglycolipids,derivatives of phosphatidylethanolamine; (3)(carbohydrate-spacer)(n)-biotin(m)-polyacrylamide. These biotinylatedderivatives may attach to lectins on the mucosal surface to facilitateabsorption of the biologically active agent(s), e.g., apolymer-encapsulated Y2 receptor-binding peptide.

[0216] Within more detailed aspects of the invention, one or more Y2receptor-binding peptide, analogs and mimetics, and/or otherbiologically active agents, disclosed herein, optionally includingsecondary active agents such as protease inhibitor(s), cytokine(s),additional modulator(s) of intercellular junctional physiology, etc.,are modified and bound to a polymeric carrier or matrix. For example,this may be accomplished by chemically binding a peptide or proteinactive agent and other optional agent(s) within a crosslinked polymernetwork. It is also possible to chemically modify the polymer separatelywith an interactive agent such as a glycosidal containing molecule. Incertain aspects, the biologically active agent(s), and optionalsecondary active agent(s), may be functionalized, i.e., wherein anappropriate reactive group is identified or is chemically added to theactive agent(s). Most often an ethylenic polymerizable group is added,and the functionalized active agent is then copolymerized with monomersand a crosslinking agent using a standard polymerization method such assolution polymerization (usually in water), emulsion, suspension ordispersion polymerization. Often, the functionalizing agent is providedwith a high enough concentration of functional or polymerizable groupsto insure that several sites on the active agent(s) are functionalized.For example, in a polypeptide comprising 16 amine sites, it is generallydesired to functionalize at least 2, 4, 5, 7, and up to 8 or more of thesites.

[0217] After functionalization, the functionalized active agent(s)is/are mixed with monomers and a crosslinking agent that comprise thereagents from which the polymer of interest is formed. Polymerization isthen induced in this medium to create a polymer containing the boundactive agent(s). The polymer is then washed with water or otherappropriate solvents and otherwise purified to remove trace unreactedimpurities and, if necessary, ground or broken up by physical means suchas by stirring, forcing it through a mesh, ultrasonication or othersuitable means to a desired particle size. The solvent, usually water,is then removed in such a manner as to not denature or otherwise degradethe active agent(s). One desired method is lyophilization (freezedrying) but other methods are available and may be used (e.g., vacuumdrying, air drying, spray drying, etc.).

[0218] To introduce polymerizable groups in peptides, proteins and otheractive agents within the invention, it is possible to react availableamino, hydroxyl, thiol and other reactive groups with electrophilescontaining unsaturated groups. For example, unsaturated monomerscontaining N-hydroxy succinimidyl groups, active carbonates such asp-nitrophenyl carbonate, trichlorophenyl carbonates, tresylate,oxycarbonylimidazoles, epoxide, isocyanates and aldehyde, andunsaturated carboxymethyl azides and unsaturated orthopyridyl-disulfidebelong to this category of reagents. Illustrative examples ofunsaturated reagents are allyl glycidyl ether, allyl chloride,allylbromide, allyl iodide, acryloyl chloride, allyl isocyanate,allylsulfonyl chloride, maleic anhydride, copolymers of maleic anhydrideand allyl ether, and the like.

[0219] All of the lysine active derivatives, except aldehyde, cangenerally react with other amino acids such as imidazole groups ofhistidine and hydroxyl groups of tyrosine and the thiol groups ofcystine if the local environment enhances nucleophilicity of thesegroups. Aldehyde containing functionalizing reagents are specific tolysine. These types of reactions with available groups from lysines,cysteines, tyrosine have been extensively documented in the literatureand are known to those skilled in the art.

[0220] In the case of biologically active agents that contain aminegroups, it is convenient to react such groups with an acyloyl chloride,such as acryloyl chloride, and introduce the polymerizable acrylic grouponto the reacted agent. Then during preparation of the polymer, such asduring the crosslinking of the copolymer of acrylamide and acrylic acid,the functionalized active agent, through the acrylic groups, is attachedto the polymer and becomes bound thereto.

[0221] In additional aspects of the invention, biologically activeagents, including peptides, proteins, nucleosides, and other moleculeswhich are bioactive in vivo, are conjugation-stabilized by covalentlybonding one or more active agent(s) to a polymer incorporating as anintegral part thereof both a hydrophilic moiety, e.g., a linearpolyalkylene glycol, a lipophilic moiety (see, e.g., U.S. Pat. No.5,681,811). In one aspect, a biologically active agent is covalentlycoupled with a polymer comprising (i) a linear polyalkylene glycolmoiety and (ii) a lipophilic moiety, wherein the active agent, linearpolyalkylene glycol moiety, and the lipophilic moiety areconformationally arranged in relation to one another such that theactive therapeutic agent has an enhanced in vivo resistance to enzymaticdegradation (i.e., relative to its stability under similar conditions inan unconjugated form devoid of the polymer coupled thereto). In anotheraspect, the conjugation-stabilized formulation has a three-dimensionalconformation comprising the biologically active agent covalently coupledwith a polysorbate complex comprising (i) a linear polyalkylene glycolmoiety and (ii) a lipophilic moiety, wherein the active agent, thelinear polyalkylene glycol moiety and the lipophilic moiety areconformationally arranged in relation to one another such that (a) thelipophilic moiety is exteriorly available in the three-dimensionalconformation, and (b) the active agent in the composition has anenhanced in vivo resistance to enzymatic degradation.

[0222] In a further related aspect, a multiligand conjugated complex isprovided which comprises a biologically active agent covalently coupledwith a triglyceride backbone moiety through a polyalkylene glycol spacergroup bonded at a carbon atom of the triglyceride backbone moiety, andat least one fatty acid moiety covalently attached either directly to acarbon atom of the triglyceride backbone moiety or covalently joinedthrough a polyalkylene glycol spacer moiety (see, e.g., U.S. Pat. No.5,681,811). In such a multiligand conjugated therapeutic agent complex,the alpha′ and beta carbon atoms of the triglyceride bioactive moietymay have fatty acid moieties attached by covalently bonding eitherdirectly thereto, or indirectly covalently bonded thereto throughpolyalkylene glycol spacer moieties. Alternatively, a fatty acid moietymay be covalently attached either directly or through a polyalkyleneglycol spacer moiety to the alpha and alpha′ carbons of the triglyceridebackbone moiety, with the bioactive therapeutic agent being covalentlycoupled with the gamma-carbon of the triglyceride backbone moiety,either being directly covalently bonded thereto or indirectly bondedthereto through a polyalkylene spacer moiety. It will be recognized thata wide variety of structural, compositional, and conformational formsare possible for the multiligand conjugated therapeutic agent complexcomprising the triglyceride backbone moiety, within the scope of theinvention. It is further noted that in such a multiligand conjugatedtherapeutic agent complex, the biologically active agent(s) mayadvantageously be covalently coupled with the triglyceride modifiedbackbone moiety through alkyl spacer groups, or alternatively otheracceptable spacer groups, within the scope of the invention. As used insuch context, acceptability of the spacer group refers to steric,compositional, and end use application specific acceptabilitycharacteristics.

[0223] In yet additional aspects of the invention, aconjugation-stabilized complex is provided which comprises a polysorbatecomplex comprising a polysorbate moiety including a triglyceridebackbone having covalently coupled to alpha, alpha′ and beta carbonatoms thereof functionalizing groups including (i) a fatty acid group;and (ii) a polyethylene glycol group having a biologically active agentor moiety covalently bonded thereto, e.g., bonded to an appropriatefunctionality of the polyethylene glycol group. Such covalent bondingmay be either direct, e.g., to a hydroxy terminal functionality of thepolyethylene glycol group, or alternatively, the covalent bonding may beindirect, e.g., by reactively capping the hydroxy terminus of thepolyethylene glycol group with a terminal carboxy functionality spacergroup, so that the resulting capped polyethylene glycol group has aterminal carboxy functionality to which the biologically active agent ormoiety may be covalently bonded.

[0224] In yet additional aspects of the invention, a stable, aqueouslysoluble, conjugation-stabilized complex is provided which comprises oneor more Y2 receptor-binding peptide proteins, analogs and mimetics,and/or other biologically active agent(s)+disclosed herein covalentlycoupled to a physiologically compatible polyethylene glycol (PEG)modified glycolipid moiety. In such complex, the biologically activeagent(s) may be covalently coupled to the physiologically compatible PEGmodified glycolipid moiety by a labile covalent bond at a free aminoacid group of the active agent, wherein the labile covalent bond isscissionable in vivo by biochemical hydrolysis and/or proteolysis. Thephysiologically compatible PEG modified glycolipid moiety mayadvantageously comprise a polysorbate polymer, e.g., a polysorbatepolymer comprising fatty acid ester groups selected from the groupconsisting of monopalmitate, dipalmitate, monolaurate, dilaurate,trilaurate, monoleate, dioleate, trioleate, monostearate, distearate,and tristearate. In such complex, the physiologically compatible PEGmodified glycolipid moiety may suitably comprise a polymer selected fromthe group consisting of polyethylene glycol ethers of fatty acids, andpolyethylene glycol esters of fatty acids, wherein the fatty acids forexample comprise a fatty acid selected from the group consisting oflauric, palmitic, oleic, and stearic acids.

[0225] Storage of Material

[0226] In certain aspects of the invention, the combinatorialformulations and/or coordinate administration methods herein incorporatean effective amount of peptides and proteins which may adhere to chargedglass thereby reducing the effective concentration in the container.Silanized containers, for example, silanized glass containers, are usedto store the finished product to reduce adsorption of the polypeptide orprotein to a glass container.

[0227] In yet additional aspects of the invention, a kit for treatmentof a mammalian subject comprises a stable pharmaceutical composition ofone or more Y2 receptor-binding peptide compound(s) formulated formucosal delivery to the mammalian subject wherein the composition iseffective to alleviate one or more symptom(s) of obesity, cancer, ormalnutrition or wasting related to cancer in said subject withoutunacceptable adverse side effects. The kit further comprises apharmaceutical reagent vial to contain the one or more Y2receptor-binding peptide compounds. The pharmaceutical reagent vial iscomposed of pharmaceutical grade polymer, glass or other suitablematerial. The pharmaceutical reagent vial is, for example, a silanizedglass vial. The kit further comprises an aperture for delivery of thecomposition to a nasal mucosal surface of the subject. The deliveryaperture is composed of a pharmaceutical grade polymer, glass or othersuitable material. The delivery aperture is, for example, a silanizedglass.

[0228] A silanization technique combines a special cleaning techniquefor the surfaces to be silanized with a silanization process at lowpressure. The silane is in the gas phase and at an enhanced temperatureof the surfaces to be silanized. The method provides reproduciblesurfaces with stable, homogeneous and functional silane layers havingcharacteristics of a monolayer. The silanized surfaces prevent bindingto the glass of polypeptides or mucosal delivery enhancing agents of thepresent invention.

[0229] The procedure is useful to prepare silanized pharmaceuticalreagent vials to hold Y2 receptor-binding peptide compositions of thepresent invention. Glass trays are cleaned by rinsing with doubledistilled water (ddH₂O) before using. The silane tray is then be rinsedwith 95% EtOH, and the acetone tray is rinsed with acetone.Pharmaceutical reagent vials are sonicated in acetone for 10 minutes.After the acetone sonication, reagent vials are washed in ddH₂O tray atleast twice. Reagent vials are sonicated in 0.1M NaOH for 10 minutes.While the reagent vials are sonicating in NaOH, the silane solution ismade under a hood. (Silane solution: 800 mL of 95% ethanol; 96 L ofglacial acetic acid; 25 mL of glycidoxypropyltrimethoxy silane). Afterthe NaOH sonication, reagent vials are washed in ddH₂O tray at leasttwice. The reagent vials are sonicated in silane solution for 3 to 5minutes. The reagent vials are washed in 100% EtOH tray. The reagentvials are dried with prepurified N₂ gas and stored in a 100° C. oven forat least 2 hours before using.

[0230] Bioadhesive Delivery Vehicles and Methods

[0231] In certain aspects of the invention, the combinatorialformulations and/or coordinate administration methods herein incorporatean effective amount of a nontoxic bioadhesive as an adjunct compound orcarrier to enhance mucosal delivery of one or more biologically activeagent(s). Bioadhesive agents in this context exhibit general or specificadhesion to one or more components or surfaces of the targeted mucosa.The bioadhesive maintains a desired concentration gradient of thebiologically active agent into or across the mucosa to ensurepenetration of even large molecules (e.g., peptides and proteins) intoor through the mucosal epithelium. Typically, employment of abioadhesive within the methods and compositions of the invention yieldsa two- to five-fold, often a five- to ten-fold increase in permeabilityfor peptides and proteins into or through the mucosal epithelium. Thisenhancement of epithelial permeation often permits effectivetransmucosal delivery of large macromolecules, for example to the basalportion of the nasal epithelium or into the adjacent extracellularcompartments or a blood plasma or CNS tissue or fluid.

[0232] This enhanced delivery provides for greatly improvedeffectiveness of delivery of bioactive peptides, proteins and othermacromolecular therapeutic species. These results will depend in part onthe hydrophilicity of the compound, whereby greater penetration will beachieved with hydrophilic species compared to water insoluble compounds.In addition to these effects, employment of bioadhesives to enhance drugpersistence at the mucosal surface can elicit a reservoir mechanism forprotracted drug delivery, whereby compounds not only penetrate acrossthe mucosal tissue but also back-diffuse toward the mucosal surface oncethe material at the surface is depleted.

[0233] A variety of suitable bioadhesives are disclosed in the art fororal administration, U.S. Pat. Nos. 3,972,995; 4,259,314; 4,680,323;4,740,365; 4,573,996; 4,292,299; 4,715,369; 4,876,092; 4,855,142;4,250,163; 4,226,848; 4,948,580; U.S. Patent Reissue 33,093, which finduse within the novel methods and compositions of the invention. Thepotential of various bioadhesive polymers as a mucosal, e.g., nasal,delivery platform within the methods and compositions of the inventioncan be readily assessed by determining their ability to retain andrelease Y2 receptor-binding peptide, as well as by their capacity tointeract with the mucosal surfaces following incorporation of the activeagent therein. In addition, well known methods will be applied todetermine the biocompatibility of selected polymers with the tissue atthe site of mucosal administration. When the target mucosa is covered bymucus (i.e., in the absence of mucolytic or mucus-clearing treatment),it can serve as a connecting link to the underlying mucosal epithelium.Therefore, the term “bioadhesive” as used herein also coversmucoadhesive compounds useful for enhancing mucosal delivery ofbiologically active agents within the invention. However, adhesivecontact to mucosal tissue mediated through adhesion to a mucus gel layermay be limited by incomplete or transient attachment between the mucuslayer and the underlying tissue, particularly at nasal surfaces whererapid mucus clearance occurs. In this regard, mucin glycoproteins arecontinuously secreted and, immediately after their release from cells orglands, form a viscoelastic gel. The luminal surface of the adherent gellayer, however, is continuously eroded by mechanical, enzymatic and/orciliary action. Where such activities are more prominent or where longeradhesion times are desired, the coordinate administration methods andcombinatorial formulation methods of the invention may furtherincorporate mucolytic and/or ciliostatic methods or agents as disclosedherein above.

[0234] Typically, mucoadhesive polymers for use within the invention arenatural or synthetic macromolecules which adhere to wet mucosal tissuesurfaces by complex, but non-specific, mechanisms. In addition to thesemucoadhesive polymers, the invention also provides methods andcompositions incorporating bioadhesives that adhere directly to a cellsurface, rather than to mucus, by means of specific, includingreceptor-mediated, interactions. One example of bioadhesives thatfunction in this specific manner is the group of compounds known aslectins. These are glycoproteins with an ability to specificallyrecognize and bind to sugar molecules, e.g. glycoproteins orglycolipids, which form part of intranasal epithelial cell membranes andcan be considered as “lectin receptors”.

[0235] In certain aspects of the invention, bioadhesive materials forenhancing intranasal delivery of biologically active agents comprise amatrix of a hydrophilic, e.g., water soluble or swellable, polymer or amixture of polymers that can adhere to a wet mucous surface. Theseadhesives may be formulated as ointments, hydrogels (see above) thinfilms, and other application forms. Often, these adhesives have thebiologically active agent mixed therewith to effectuate slow release orlocal delivery of the active agent. Some are formulated with additionalingredients to facilitate penetration of the active agent through thenasal mucosa, e.g., into the circulatory system of the individual.

[0236] Various polymers, both natural and synthetic ones, showsignificant binding to mucus and/or mucosal epithelial surfaces underphysiological conditions. The strength of this interaction can readilybe measured by mechanical peel or shear tests. When applied to a humidmucosal surface, many dry materials will spontaneously adhere, at leastslightly. After such an initial contact, some hydrophilic materialsstart to attract water by adsorption, swelling or capillary forces, andif this water is absorbed from the underlying substrate or from thepolymer-tissue interface, the adhesion may be sufficient to achieve thegoal of enhancing mucosal absorption of biologically active agents. Such‘adhesion by hydration’ can be quite strong, but formulations adapted toemploy this mechanism must account for swelling which continues as thedosage transforms into a hydrated mucilage. This is projected for manyhydrocolloids useful within the invention, especially somecellulose-derivatives, which are generally non-adhesive when applied inpre-hydrated state. Nevertheless, bioadhesive drug delivery systems formucosal administration are effective within the invention when suchmaterials are applied in the form of a dry polymeric powder,microsphere, or film-type delivery form.

[0237] Other polymers adhere to mucosal surfaces not only when appliedin dry, but also in fully hydrated state, and in the presence of excessamounts of water. The selection of a mucoadhesive thus requires dueconsideration of the conditions, physiological as well asphysico-chemical, under which the contact to the tissue will be formedand maintained. In particular, the amount of water or humidity usuallypresent at the intended site of adhesion, and the prevailing pH, areknown to largely affect the mucoadhesive binding strength of differentpolymers.

[0238] Several polymeric bioadhesive drug delivery systems have beenfabricated and studied in the past 20 years, not always with success. Avariety of such carriers are, however, currently used in clinicalapplications involving dental, orthopedic, ophthalmological, andsurgical uses. For example, acrylic-based hydrogels have been usedextensively for bioadhesive devices. Acrylic-based hydrogels are wellsuited for bioadhesion due to their flexibility and nonabrasivecharacteristics in the partially swollen state, which reducedamage-causing attrition to the tissues in contact. Furthermore, theirhigh permeability in the swollen state allows unreacted monomer,un-crosslinked polymer chains, and the initiator to be washed out of thematrix after polymerization, which is an important feature for selectionof bioadhesive materials for use within the invention. Acrylic-basedpolymer devices exhibit very high adhesive bond strength. For controlledmucosal delivery of peptide and protein drugs, the methods andcompositions of the invention optionally include the use of carriers,e.g., polymeric delivery vehicles, that function in part to shield thebiologically active agent from proteolytic breakdown, while at the sametime providing for enhanced penetration of the peptide or protein intoor through the nasal mucosa. In this context, bioadhesive polymers havedemonstrated considerable potential for enhancing oral drug delivery. Asan example, the bioavailability of 9-desglycinamide, 8-argininevasopressin (DGAVP) intraduodenally administered to rats together with a1% (w/v) saline dispersion of the mucoadhesive poly(acrylic acid)derivative polycarbophil, was 3-5-fold increased compared to an aqueoussolution of the peptide drug without this polymer.

[0239] Mucoadhesive polymers of the poly(acrylic acid)-type are potentinhibitors of some intestinal proteases. The mechanism of enzymeinhibition is explained by the strong affinity of this class of polymersfor divalent cations, such as calcium or zinc, which are essentialcofactors of metallo-proteinases, such as trypsin and chymotrypsin.Depriving the proteases of their cofactors by poly(acrylic acid) wasreported to induce irreversible structural changes of the enzymeproteins which were accompanied by a loss of enzyme activity. At thesame time, other mucoadhesive polymers (e.g., some cellulose derivativesand chitosan) may not inhibit proteolytic enzymes under certainconditions. In contrast to other enzyme inhibitors contemplated for usewithin the invention (e.g. aprotinin, bestatin), which are relativelysmall molecules, the trans-nasal absorption of inhibitory polymers islikely to be minimal in light of the size of these molecules, andthereby eliminate possible adverse side effects. Thus, mucoadhesivepolymers, particularly of the poly(acrylic acid)-type, may serve both asan absorption-promoting adhesive and enzyme-protective agent to enhancecontrolled delivery of peptide and protein drugs, especially when safetyconcerns are considered.

[0240] In addition to protecting against enzymatic degradation,bioadhesives and other polymeric or non-polymeric absorption-promotingagents for use within the invention may directly increase mucosalpermeability to biologically active agents. To facilitate the transportof large and hydrophilic molecules, such as peptides and proteins,across the nasal epithelial barrier, mucoadhesive polymers and otheragents have been postulated to yield enhanced permeation effects beyondwhat is accounted for by prolonged premucosal residence time of thedelivery system. The time course of drug plasma concentrationsreportedly suggested that the bioadhesive microspheres caused an acute,but transient increase of insulin permeability across the nasal mucosa.Other mucoadhesive polymers for use within the invention, for examplechitosan, reportedly enhance the permeability of certain mucosalepithelia even when they are applied as an aqueous solution or gel.Another mucoadhesive polymer reported to directly affect epithelialpermeability is hyaluronic acid and ester derivatives thereof. Aparticularly useful bioadhesive agent within the coordinateadministration, and/or combinatorial formulation methods andcompositions of the invention is chitosan, as well as its analogs andderivatives. Chitosan is a non-toxic, biocompatible and biodegradablepolymer that is widely used for pharmaceutical and medical applicationsbecause of its favorable properties of low toxicity and goodbiocompatibility. It is a natural polyaminosaccharide prepared fromchitin by N-deacetylation with alkali. As used within the methods andcompositions of the invention, chitosan increases the retention of Y2receptor-binding peptide proteins, analogs and mimetics, and otherbiologically active agents disclosed herein at a mucosal site ofapplication. This mode of administration can also improve patientcompliance and acceptance. As further provided herein, the methods andcompositions of the invention will optionally include a novel chitosanderivative or chemically modified form of chitosan. One such novelderivative for use within the invention is denoted as aβ-[1→4]-2-guanidino-2-deoxy-D-glucose polymer (poly-GuD). Chitosan isthe N-deacetylated product of chitin, a naturally occurring polymer thathas been used extensively to prepare microspheres for oral andintra-nasal formulations. The chitosan polymer has also been proposed asa soluble carrier for parenteral drug delivery. Within one aspect of theinvention, o-methylisourea is used to convert a chitosan amine to itsguanidinium moiety. The guanidinium compound is prepared, for example,by the reaction between equi-normal solutions of chitosan ando-methylisourea at pH above 8.0.

[0241] The guanidinium product is -[14]-guanidino-2-deoxy-D-glucosepolymer. It is abbreviated as Poly-GuD in this context (Monomer F. W. ofAmine in Chitosan=161; Monomer F. W. of Guanidinium in Poly-GuD=203).

[0242] One exemplary Poly-GuD preparation method for use within theinvention involves the following protocol.

[0243] Solutions:

[0244] Preparation of 0.5% Acetic Acid Solution (0.088N):

[0245] Pipette 2.5 mL glacial acetic acid into a 500 mL volumetricflask, dilute to volume with purified water.

[0246] Preparation of 2N NaOH Solution:

[0247] Transfer about 20 g NaOH pellets into a beaker with about 150 mLof purified water. Dissolve and cool to room temperature. Transfer thesolution into a 250-mL volumetric flask, dilute to volume with purifiedwater.

[0248] Preparation of O-methylisourea Sulfate (0.4N urea groupequivalent):

[0249] Transfer about 493 mg of O-methylisourea sulfate into a 10-mLvolumetric flask, dissolve and dilute to volume with purified water.

[0250] The pH of the solution is 4.2

[0251] Preparation of Barium Chloride Solution (0.2M):

[0252] Transfer about 2.086 g of Barium chloride into a 50-mL volumetricflask, dissolve and dilute to volume with purified water.

[0253] Preparation of Chitosan Solution (0.06N Amine Equivalent):

[0254] Transfer about 100 mg Chitosan into a 50 mL beaker, add 10 mL0.5% Acetic Acid (0.088 N). Stir to dissolve completely.

[0255] The pH of the solution is about 4.5

[0256] Preparation of O-methylisourea Chloride Solution (0.2N Urea GroupEquivalent):

[0257] Pipette 5.0 mL of O-methylisourea sulfate solution (0.4 N ureagroup equivalent) and 5 mL of 0.2M Barium chloride solution into abeaker. A precipitate is formed. Continue to mix the solution foradditional 5 minutes. Filter the solution through 0.45 m filter anddiscard the precipitate. The concentration of O-methylisourea chloridein the supernatant solution is 0.2 N urea group equivalents.

[0258] The pH of the solution is 4.2.

[0259] Procedure:

[0260] Add 1.5 mL of 2 N NaOH to 10 mL of the chitosan solution (0.06Namine equivalent) prepared as described in Section 2.5. Adjust the pH ofthe solution with 2N NaOH to about 8.2 to 8.4. Stir the solution foradditional 10 minutes. Add 3.0 mL O-methylisourea chloride solution(0.2N urea group equivalent) prepared as described above. Stir thesolution overnight.

[0261] Adjust the pH of solution to 5.5 with 0.5% Acetic Acid (0.088N).

[0262] Dilute the solution to a final volume of 25 mL using purifiedwater.

[0263] The Poly-GuD concentration in the solution is 5 mg/mL, equivalentto 0.025 N (guanidium group).

[0264] Additional compounds classified as bioadhesive agents for usewithin the present invention act by mediating specific interactions,typically classified as “receptor-ligand interactions” betweencomplementary structures of the bioadhesive compound and a component ofthe mucosal epithelial surface. Many natural examples illustrate thisform of specific binding bioadhesion, as exemplified by lectin-sugarinteractions. Lectins are (glyco) proteins of non-immune origin whichbind to polysaccharides or glycoconjugates.

[0265] Several plant lectins have been investigated as possiblepharmaceutical absorption-promoting agents. One plant lectin, Phaseolusvulgaris hemagglutinin (PHA), exhibits high oral bioavailability of morethan 10% after feeding to rats. Tomato (Lycopersicon esculeutum) lectin(TL) appears safe for various modes of administration.

[0266] In summary, the foregoing bioadhesive agents are useful in thecombinatorial formulations and coordinate administration methods of theinstant invention, which optionally incorporate an effective amount andform of a bioadhesive agent to prolong persistence or otherwise increasemucosal absorption of one or more Y2 receptor-binding peptide proteins,analogs and mimetics, and other biologically active agents. Thebioadhesive agents may be coordinately administered as adjunct compoundsor as additives within the combinatorial formulations of the invention.In certain embodiments, the bioadhesive agent acts as a ‘pharmaceuticalglue’, whereas in other embodiments adjunct delivery or combinatorialformulation of the bioadhesive agent serves to intensify contact of thebiologically active agent with the nasal mucosa, in some cases bypromoting specific receptor-ligand interactions with epithelial cell“receptors”, and in others by increasing epithelial permeability tosignificantly increase the drug concentration gradient measured at atarget site of delivery (e.g., liver, blood plasma, or CNS tissue orfluid). Yet additional bioadhesive agents for use within the inventionact as enzyme (e.g., protease) inhibitors to enhance the stability ofmucosally administered biotherapeutic agents delivered coordinately orin a combinatorial formulation with the bioadhesive agent.

[0267] Liposomes and Micellar Delivery Vehicles

[0268] The coordinate administration methods and combinatorialformulations of the instant invention optionally incorporate effectivelipid or fatty acid based carriers, processing agents, or deliveryvehicles, to provide improved formulations for mucosal delivery of Y2receptor-binding peptide proteins, analogs and mimetics, and otherbiologically active agents. For example, a variety of formulations andmethods are provided for mucosal delivery which comprise one or more ofthese active agents, such as a peptide or protein, admixed orencapsulated by, or coordinately administered with, a liposome, mixedmicellar carrier, or emulsion, to enhance chemical and physicalstability and increase the half life of the biologically active agents(e.g., by reducing susceptibility to proteolysis, chemical modificationand/or denaturation) upon mucosal delivery.

[0269] Within certain aspects of the invention, specialized deliverysystems for biologically active agents comprise small lipid vesiclesknown as liposomes. These are typically made from natural,biodegradable, non-toxic, and non-immunogenic lipid molecules, and canefficiently entrap or bind drug molecules, including peptides andproteins, into, or onto, their membranes. The attractiveness ofliposomes as a peptide and protein delivery system within the inventionis increased by the fact that the encapsulated proteins can remain intheir preferred aqueous environment within the vesicles, while theliposomal membrane protects them against proteolysis and otherdestabilizing factors. Even though not all liposome preparation methodsknown are feasible in the encapsulation of peptides and proteins due totheir unique physical and chemical properties, several methods allow theencapsulation of these macromolecules without substantial deactivation.

[0270] A variety of methods are available for preparing liposomes foruse within the invention, U.S. Pat. Nos. 4,235,871, 4,501,728, and4,837,028. For use with liposome delivery, the biologically active agentis typically entrapped within the liposome, or lipid vesicle, or isbound to the outside of the vesicle.

[0271] Like liposomes, unsaturated long chain fatty acids, which alsohave enhancing activity for mucosal absorption, can form closed vesicleswith bilayer-like structures (so called “ufasomes”). These can beformed, for example, using oleic acid to entrap biologically activepeptides and proteins for mucosal, e.g., intranasal, delivery within theinvention.

[0272] Other delivery systems for use within the invention combine theuse of polymers and liposomes to ally the advantageous properties ofboth vehicles such as encapsulation inside the natural polymer fibrin.In addition, release of biotherapeutic compounds from this deliverysystem is controllable through the use of covalent crosslinking and theaddition of antifibrinolytic agents to the fibrin polymer.

[0273] More simplified delivery systems for use within the inventioninclude the use of cationic lipids as delivery vehicles or carriers,which can be effectively employed to provide an electrostaticinteraction between the lipid carrier and such charged biologicallyactive agents as proteins and polyanionic nucleic acids. This allowsefficient packaging of the drugs into a form suitable for mucosaladministration and/or subsequent delivery to systemic compartments.

[0274] Additional delivery vehicles for use within the invention includelong and medium chain fatty acids, as well as surfactant mixed micelleswith fatty acids. Most naturally occurring lipids in the form of estershave important implications with regard to their own transport acrossmucosal surfaces. Free fatty acids and their monoglycerides which havepolar groups attached have been demonstrated in the form of mixedmicelles to act on the intestinal barrier as penetration enhancers. Thisdiscovery of barrier modifying function of free fatty acids (carboxylicacids with a chain length varying from 12 to 20 carbon atoms) and theirpolar derivatives has stimulated extensive research on the applicationof these agents as mucosal absorption enhancers.

[0275] For use within the methods of the invention, long chain fattyacids, especially fusogenic lipids (unsaturated fatty acids andmonoglycerides such as oleic acid, linoleic acid, linoleic acid,monoolein, etc.) provide useful carriers to enhance mucosal delivery ofY2 receptor-binding peptide, analogs and mimetics, and otherbiologically active agents disclosed herein. Medium chain fatty acids(C6 to C12) and monoglycerides have also been shown to have enhancingactivity in intestinal drug absorption and can be adapted for use withinthe mocosal delivery formulations and methods of the invention. Inaddition, sodium salts of medium and long chain fatty acids areeffective delivery vehicles and absorption-enhancing agents for mucosaldelivery of biologically active agents within the invention. Thus, fattyacids can be employed in soluble forms of sodium salts or by theaddition of non-toxic surfactants, e.g., polyoxyethylated hydrogenatedcastor oil, sodium taurocholate, etc. Other fatty acid and mixedmicellar preparations that are useful within the invention include, butare not limited to, Na caprylate (C8), Na caprate (C 10), Na laurate (C12) or Na oleate (C 18), optionally combined with bile salts, such asglycocholate and taurocholate.

[0276] Pegylation

[0277] Additional methods and compositions provided within the inventioninvolve chemical modification of biologically active peptides andproteins by covalent attachment of polymeric materials, for exampledextrans, polyvinyl pyrrolidones, glycopeptides, polyethylene glycol andpolyamino acids. The resulting conjugated peptides and proteins retaintheir biological activities and solubility for mucosal administration.In alternate embodiments, Y2 receptor-binding peptide proteins, analogsand mimetics, and other biologically active peptides and proteins, areconjugated to polyalkylene oxide polymers, particularly polyethyleneglycols (PEG). U.S. Pat. No. 4,179,337.

[0278] Amine-reactive PEG polymers for use within the invention includeSC-PEG with molecular masses of 2000, 5000, 10000, 12000, and 20000;U-PEG-10000; NHS-PEG-3400-biotin; T-PEG-5000; T-PEG-12000; andTPC-PEG-5000. PEGylation of biologically active peptides and proteinsmay be achieved by modification of carboxyl sites (e.g., aspartic acidor glutamic acid groups in addition to the carboxyl terminus). Theutility of PEG-hydrazide in selective modification ofcarbodiimide-activated protein carboxyl groups under acidic conditionshas been described. Alternatively, bifunctional PEG modification ofbiologically active peptides and proteins can be employed. In someprocedures, charged amino acid residues, including lysine, asparticacid, and glutamic acid, have a marked tendency to be solvent accessibleon protein surfaces.

[0279] Other Stabilizing Modifications of Active Agents

[0280] In addition to PEGylation, biologically active agents such aspeptides and proteins for use within the invention can be modified toenhance circulating half-life by shielding the active agent viaconjugation to other known protecting or stabilizing compounds, forexample by the creation of fusion proteins with an active peptide,protein, analog or mimetic linked to one or more carrier proteins, suchas one or more immunoglobulin chains.

[0281] Formulation and Administration

[0282] Mucosal delivery formulations of the present invention compriseY2 receptor-binding peptide, analogs and mimetics, typically combinedtogether with one or more pharmaceutically acceptable carriers and,optionally, other therapeutic ingredients. The carrier(s) must be“pharmaceutically acceptable” in the sense of being compatible with theother ingredients of the formulation and not eliciting an unacceptabledeleterious effect in the subject. Such carriers are described hereinabove or are otherwise well known to those skilled in the art ofpharmacology. Desirably, the formulation should not include substancessuch as enzymes or oxidizing agents with which the biologically activeagent to be administered is known to be incompatible. The formulationsmay be prepared by any of the methods well known in the art of pharmacy.

[0283] Within the compositions and methods of the invention, the Y2receptor-binding peptide proteins, analogs and mimetics, and otherbiologically active agents disclosed herein may be administered tosubjects by a variety of mucosal administration modes, including byoral, rectal, vaginal, intranasal, intrapulmonary, or transdermaldelivery, or by topical delivery to the eyes, ears, skin or othermucosal surfaces. Optionally, Y2 receptor-binding peptide proteins,analogs and mimetics, and other biologically active agents disclosedherein can be coordinately or adjunctively administered by non-mucosalroutes, including by intramuscular, subcutaneous, intravenous,intra-atrial, intra-articular, intraperitoneal, or parenteral routes. Inother alternative embodiments, the biologically active agent(s) can beadministered ex vivo by direct exposure to cells, tissues or organsoriginating from a mammalian subject, for example as a component of anex vivo tissue or organ treatment formulation that contains thebiologically active agent in a suitable, liquid or solid carrier.

[0284] Compositions according to the present invention are oftenadministered in an aqueous solution as a nasal or pulmonary spray andmay be dispensed in spray form by a variety of methods known to thoseskilled in the art. Preferred systems for dispensing liquids as a nasalspray are disclosed in U.S. Pat. No. 4,511,069. The formulations may bepresented in multi-dose containers, for example in the sealed dispensingsystem disclosed in U.S. Pat. No. 4,511,069. Additional aerosol deliveryforms may include, e.g., compressed air-, jet-, ultrasonic-, andpiezoelectric nebulizers, which deliver the biologically active agentdissolved or suspended in a pharmaceutical solvent, e.g., water,ethanol, or a mixture thereof.

[0285] Nasal and pulmonary spray solutions of the present inventiontypically comprise the drug or drug to be delivered, optionallyformulated with a surface-active agent, such as a nonionic surfactant(e.g., polysorbate-80), and one or more buffers. In some embodiments ofthe present invention, the nasal spray solution further comprises apropellant. The pH of the nasal spray solution is optionally betweenabout pH 3.0 and 6.0, preferably 5.0±0.3. Suitable buffers for usewithin these compositions are as described above or as otherwise knownin the art. Other components may be added to enhance or maintainchemical stability, including preservatives, surfactants, dispersants,or gases. Suitable preservatives include, but are not limited to,phenol, methyl paraben, paraben, m-cresol, thiomersal, chlorobutanol,benzylalkonimum chloride, and the like. Suitable surfactants include,but are not limited to, oleic acid, sorbitan trioleate, polysorbates,lecithin, phosphotidyl cholines, and various long chain diglycerides andphospholipids. Suitable dispersants include, but are not limited to,ethylenediaminetetraacetic acid, and the like. Suitable gases include,but are not limited to, nitrogen, helium, chlorofluorocarbons (CFCs),hydrofluorocarbons (HFCs), carbon dioxide, air, and the like.

[0286] Within alternate embodiments, mucosal formulations areadministered as dry powder formulations comprising the biologicallyactive agent in a dry, usually lyophilized, form of an appropriateparticle size, or within an appropriate particle size range, forintranasal delivery. Minimum particle size appropriate for depositionwithin the nasal or pulmonary passages is often about 0.5μ mass medianequivalent aerodynamic diameter (MMEAD), commonly about 1μ MMEAD, andmore typically about 2μ MMEAD. Maximum particle size appropriate fordeposition within the nasal passages is often about 10μ MMEAD, commonlyabout 8μ MMEAD, and more typically about 4μ MMEAD. Intranasallyrespirable powders within these size ranges can be produced by a varietyof conventional techniques, such as jet milling, spray drying, solventprecipitation, supercritical fluid condensation, and the like. These drypowders of appropriate MMEAD can be administered to a patient via aconventional dry powder inhaler (DPI), which rely on the patient'sbreath, upon pulmonary or nasal inhalation, to disperse the power intoan aerosolized amount. Alternatively, the dry powder may be administeredvia air-assisted devices that use an external power source to dispersethe powder into an aerosolized amount, e.g., a piston pump.

[0287] Dry powder devices typically require a powder mass in the rangefrom about 1 mg to 20 mg to produce a single aerosolized dose (“puff”).If the required or desired dose of the biologically active agent islower than this amount, the powdered active agent will typically becombined with a pharmaceutical dry bulking powder to provide therequired total powder mass. Preferred dry bulking powders includesucrose, lactose, dextrose, mannitol, glycine, trehalose, human serumalbumin (HSA), and starch. Other suitable dry bulking powders includecellobiose, dextrans, maltotriose, pectin, sodium citrate, sodiumascorbate, and the like.

[0288] To formulate compositions for mucosal delivery within the presentinvention, the biologically active agent can be combined with variouspharmaceutically acceptable additives, as well as a base or carrier fordispersion of the active agent(s). Desired additives include, but arenot limited to, pH control agents, such as arginine, sodium hydroxide,glycine, hydrochloric acid, citric acid, etc. In addition, localanesthetics (e.g., benzyl alcohol), isotonizing agents (e.g., sodiumchloride, mannitol, sorbitol), adsorption inhibitors (e.g., Tween 80),solubility enhancing agents (e.g., cyclodextrins and derivativesthereof), stabilizers (e.g., serum albumin), and reducing agents (e.g.,glutathione) can be included. When the composition for mucosal deliveryis a liquid, the tonicity of the formulation, as measured with referenceto the tonicity of 0.9% (w/v) physiological saline solution taken asunity, is typically adjusted to a value at which no substantial,irreversible tissue damage will be induced in the nasal mucosa at thesite of administration. Generally, the tonicity of the solution isadjusted to a value of about ⅓ to 3, more typically ½ to 2, and mostoften ¾ to 1.7.

[0289] The biologically active agent may be dispersed in a base orvehicle, which may comprise a hydrophilic compound having a capacity todisperse the active agent and any desired additives. The base may beselected from a wide range of suitable carriers, including but notlimited to, copolymers of polycarboxylic acids or salts thereof,carboxylic anhydrides (e.g. maleic anhydride) with other monomers (e.g.methyl (meth)acrylate, acrylic acid, etc.), hydrophilic vinyl polymerssuch as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone,cellulose derivatives such as hydroxymethylcellulose,hydroxypropylcellulose, etc., and natural polymers such as chitosan,collagen, sodium alginate, gelatin, hyaluronic acid, and nontoxic metalsalts thereof. Often, a biodegradable polymer is selected as a base orcarrier, for example, polylactic acid, poly(lactic acid-glycolic acid)copolymer, polyhydroxybutyric acid, poly(hydroxybutyric acid-glycolicacid) copolymer and mixtures thereof. Alternatively or additionally,synthetic fatty acid esters such as polyglycerin fatty acid esters,sucrose fatty acid esters, etc. can be employed as carriers. Hydrophilicpolymers and other carriers can be used alone or in combination, andenhanced structural integrity can be imparted to the carrier by partialcrystallization, ionic bonding, crosslinking and the like. The carriercan be provided in a variety of forms, including, fluid or viscoussolutions, gels, pastes, powders, microspheres and films for directapplication to the nasal mucosa. The use of a selected carrier in thiscontext may result in promotion of absorption of the biologically activeagent.

[0290] The biologically active agent can be combined with the base orcarrier according to a variety of methods, and release of the activeagent may be by diffusion, disintegration of the carrier, or associatedformulation of water channels. In some circumstances, the active agentis dispersed in microcapsules (microspheres) or nanocapsules(nanospheres) prepared from a suitable polymer, e.g., isobutyl2-cyanoacrylate and dispersed in a biocompatible dispersing mediumapplied to the nasal mucosa, which yields sustained delivery andbiological activity over a protracted time.

[0291] To further enhance mucosal delivery of pharmaceutical agentswithin the invention, formulations comprising the active agent may alsocontain a hydrophilic low molecular weight compound as a base orexcipient. Such hydrophilic low molecular weight compounds provide apassage medium through which a water-soluble active agent, such as aphysiologically active peptide or protein, may diffuse through the baseto the body surface where the active agent is absorbed. The hydrophiliclow molecular weight compound optionally absorbs moisture from themucosa or the administration atmosphere and dissolves the water-solubleactive peptide. The molecular weight of the hydrophilic low molecularweight compound is generally not more than 10000 and preferably not morethan 3000. Exemplary hydrophilic low molecular weight compound includepolyol compounds, such as oligo-, di- and monosaccarides such assucrose, mannitol, sorbitol, lactose, L-arabinose, D-erythrose,D-ribose, D-xylose, D-mannose, trehalose, D-galactose, lactulose,cellobiose, gentibiose, glycerin and polyethylene glycol. Other examplesof hydrophilic low molecular weight compounds useful as carriers withinthe invention include N-methylpyrrolidone, and alcohols (e.g. oligovinylalcohol, ethanol, ethylene glycol, propylene glycol, etc.) Thesehydrophilic low molecular weight compounds can be used alone or incombination with one another or with other active or inactive componentsof the intranasal formulation.

[0292] The compositions of the invention may alternatively contain aspharmaceutically acceptable carriers substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, triethanolamineoleate, etc. For solid compositions, conventional nontoxicpharmaceutically acceptable carriers can be used which include, forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharin, talcum, cellulose, glucose, sucrose,magnesium carbonate, and the like.

[0293] Therapeutic compositions for administering the biologicallyactive agent can also be formulated as a solution, microemulsion, orother ordered structure suitable for high concentration of activeingredients. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like), andsuitable mixtures thereof. Proper fluidity for solutions can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of a desired particle size in the case of dispersibleformulations, and by the use of surfactants. In many cases, it will bedesirable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, or sodium chloride in the composition.Prolonged absorption of the biologically active agent can be broughtabout by including in the composition an agent which delays absorption,for example, monostearate salts and gelatin.

[0294] In certain embodiments of the invention, the biologically activeagent is administered in a time-release formulation, for example in acomposition which includes a slow release polymer. The active agent canbe prepared with carriers that will protect against rapid release, forexample a controlled release vehicle such as a polymer,microencapsulated delivery system or bioadhesive gel. Prolonged deliveryof the active agent, in various compositions of the invention can bebrought about by including in the composition agents that delayabsorption, for example, aluminum monosterate hydrogels and gelatin.When controlled release formulations of the biologically active agent isdesired, controlled release binders suitable for use in accordance withthe invention include any biocompatible controlled-release materialwhich is inert to the active agent and which is capable of incorporatingthe biologically active agent. Numerous such materials are known in theart. Useful controlled-release binders are materials that aremetabolized slowly under physiological conditions following theirintranasal delivery (e.g., at the nasal mucosal surface, or in thepresence of bodily fluids following transmucosal delivery). Appropriatebinders include but are not limited to biocompatible polymers andcopolymers previously used in the art in sustained release formulations.Such biocompatible compounds are non-toxic and inert to surroundingtissues, and do not trigger significant adverse side effects such asnasal irritation, immune response, inflammation, or the like. They aremetabolized into metabolic products that are also biocompatible andeasily eliminated from the body.

[0295] Exemplary polymeric materials for use in this context include,but are not limited to, polymeric matrices derived from copolymeric andhomopolymeric polyesters having hydrolysable ester linkages. A number ofthese are known in the art to be biodegradable and to lead todegradation products having no or low toxicity. Exemplary polymersinclude polyglycolic acids (PGA) and polylactic acids (PLA),poly(DL-lactic acid-co-glycolic acid)(DL PLGA), poly(D-lacticacid-coglycolic acid)(D PLGA) and poly(L-lactic acid-co-glycolic acid)(LPLGA). Other useful biodegradable or bioerodable polymers include butare not limited to such polymers as poly(epsilon-caprolactone),poly(epsilon-aprolactone-CO-lactic acid), poly(E-aprolactone-CO-glycolicacid), poly(beta-hydroxy butyric acid), poly(alkyl-2-cyanoacrilate),hydrogels such as poly(hydroxyethyl methacrylate), polyamides,poly(amino acids) (i.e., L-leucine, glutamic acid, L-aspartic acid andthe like), poly (ester urea), poly (2-hydroxyethyl DL-aspartamide),polyacetal polymers, polyorthoesters, polycarbonate, polymaleamides,polysaccharides and copolymers thereof. Many methods for preparing suchformulations are generally known to those skilled in the art. Otheruseful formulations include controlled-release compositions e.g.,microcapsules, U.S. Pat. Nos. 4,652,441 and 4,917,893, lacticacid-glycolic acid copolymers useful in making microcapsules and otherformulations, U.S. Pat. Nos. 4,677,191 and 4,728,721, andsustained-release compositions for water-soluble peptides, U.S. Pat. No.4,675,189.

[0296] Sterile solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders, methods of preparationinclude vacuum drying and freeze-drying which yields a powder of theactive ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof. The prevention of theaction of microorganisms can be accomplished by various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol,sorbic acid, thimerosal, and the like.

[0297] Mucosal administration according to the invention allowseffective self-administration of treatment by patients, provided thatsufficient safeguards are in place to control and monitor dosing andside effects. Mucosal administration also overcomes certain drawbacks ofother administration forms, such as injections, that are painful andexpose the patient to possible infections and may present drugbioavailability problems. For nasal and pulmonary delivery, systems forcontrolled aerosol dispensing of therapeutic liquids as a spray are wellknown. In one embodiment, metered doses of active agent are delivered bymeans of a specially constructed mechanical pump valve, U.S. Pat. No.4,511,069.

[0298] Dosage

[0299] For prophylactic and treatment purposes, the biologically activeagent(s) disclosed herein may be administered to the subject in a singlebolus delivery, via continuous delivery (e.g., continuous transdermal,mucosal, or intravenous delivery) over an extended time period, or in arepeated administration protocol (e.g., by an hourly, daily or weekly,repeated administration protocol). In this context, a therapeuticallyeffective dosage of the Y2 receptor-binding peptide may include repeateddoses within a prolonged prophylaxis or treatment regimen that willyield clinically significant results to alleviate one or more symptomsor detectable conditions associated with a targeted disease or conditionas set forth above. Determination of effective dosages in this contextis typically based on animal model studies followed up by human clinicaltrials and is guided by determining effective dosages and administrationprotocols that significantly reduce the occurrence or severity oftargeted disease symptoms or conditions in the subject. Suitable modelsin this regard include, for example, murine, rat, porcine, feline,non-human primate, and other accepted animal model subjects known in theart. Alternatively, effective dosages can be determined using in vitromodels (e.g., immunologic and histopathologic assays). Using suchmodels, only ordinary calculations and adjustments are typicallyrequired to determine an appropriate concentration and dose toadminister a therapeutically effective amount of the biologically activeagent(s) (e.g., amounts that are intranasally effective, transdermallyeffective, intravenously effective, or intramuscularly effective toelicit a desired response).

[0300] The actual dosage of biologically active agents will of coursevary according to factors such as the disease indication and particularstatus of the subject (e.g., the subject's age, size, fitness, extent ofsymptoms, susceptibility factors, etc), time and route ofadministration, other drugs or treatments being administeredconcurrently, as well as the specific pharmacology of the biologicallyactive agent(s) for eliciting the desired activity or biologicalresponse in the subject. Dosage regimens may be adjusted to provide anoptimum prophylactic or therapeutic response. A therapeuticallyeffective amount is also one in which any toxic or detrimental sideeffects of the biologically active agent are outweighed in clinicalterms by therapeutically beneficial effects. A non-limiting range for atherapeutically effective amount of an Y2 agonist within the methods andformulations of the invention is 0.7 μg/kg to about 25 μg/kg. To promoteweight loss, an intranasal dose of Y2 receptor-binding peptide isadministered at dose high enough to promote satiety but low enough so asnot to induce any unwanted side-effects such as nausea. A preferredintranasal dose of PYY₃₋₃₆ is about 1 μg-10 μg/kg weight of the patient,most preferably from about 1.5 μg/kg to about 3 μg/kg weight of thepatient. In a standard dose a patient will receive 50 μg to 1600 μg,more preferably about between 75 μg to 800 μg, most preferably 100 μg,150 μg, 200 μg to about 400 μg. Alternatively, a non-limiting range fora therapeutically effective amount of a biologically active agent withinthe methods and formulations of the invention is between about 0.001pmol to about 100 pmol per kg body weight, between about 0.01 pmol toabout 10 pmol per kg body weight, between about 0.1 pmol to about 5 pmolper kg body weight, or between about 0.5 pmol to about 1.0 pmol per kgbody weight. Dosages within this range can be achieved by single ormultiple administrations, including, e.g., multiple administrations perday, daily or weekly administrations. Per administration, it isdesirable to administer at least one microgram of the biologicallyactive agent (e.g., one or more Y2 receptor-binding peptide proteins,analogs and mimetics, and other biologically active agents), moretypically between about 10 μg and 5.0 mg, and in certain embodimentsbetween about 100 μg and 1.0 or 2.0 mg to an average human subject. Forcertain oral applications, doses as high as 0.5 mg per kg body weightmay be necessary to achieve adequate plasma levels. It is to be furthernoted that for each particular subject, specific dosage regimens shouldbe evaluated and adjusted over time according to the individual need andprofessional judgment of the person administering or supervising theadministration of the permeabilizing peptide(s) and other biologicallyactive agent(s). An intranasal dose of a PYY will range from 50 μg to1600 μg of PYY, preferably 75 μg to 800 μg, more preferably 100 μg to400 μg with a most preferred dose being between 100 μg to 200 μg with150 μg being a dose that is considered to be highly effective. Repeatedintranasal dosing with the formulations of the invention, on a scheduleranging from about 0.1 to 24 hours between doses, preferably between 0.5and 24.0 hours between doses, will maintain normalized, sustainedtherapeutic levels of Y2 receptor-binding peptide to maximize clinicalbenefits while minimizing the risks of excessive exposure and sideeffects. This dose can be administered several times a day to promotesatiety, preferably one half hour before a meal or when hunger occurs.The goal is to mucosally deliver an amount of the Y2 receptor-bindingpeptide sufficient to raise the concentration of the Y2 receptor-bindingpeptide in the plasma of an individual to mimic the concentration thatwould normally occur postpradially, i.e., after the individual hasfinished eating.

[0301] Dosage of Y2 agonists such as PYY may be varied by the attendingclinician or patient, if self administering an over the counter dosageform, to maintain a desired concentration at the target site.

[0302] In an alternative embodiment, the invention provides compositionsand methods for intranasal delivery of Y2 receptor-binding peptide,wherein the Y2 receptor-binding peptide compound(s) is/are repeatedlyadministered through an intranasal effective dosage regimen thatinvolves multiple administrations of the Y2 receptor-binding peptide tothe subject during a daily or weekly schedule to maintain atherapeutically effective elevated and lowered pulsatile level of Y2receptor-binding peptide during an extended dosing period. Thecompositions and method provide Y2 receptor-binding peptide compound(s)that are self-administered by the subject in a nasal formulation betweenone and six times daily to maintain a therapeutically effective elevatedand lowered pulsatile level of Y2 receptor-binding peptide during an 8hour to 24 hour extended dosing period.

[0303] Kits

[0304] The instant invention also includes kits, packages andmulticontainer units containing the above described pharmaceuticalcompositions, active ingredients, and/or means for administering thesame for use in the prevention and treatment of diseases and otherconditions in mammalian subjects. Briefly, these kits include acontainer or formulation that contains one or more Y2 receptor-bindingpeptide proteins, analogs or mimetics, and/or other biologically activeagents in combination with mucosal delivery enhancing agents disclosedherein formulated in a pharmaceutical preparation for mucosal delivery.

[0305] The intranasal formulations of the present invention can beadministered using any spray bottle or syringe. An example of a nasalspray bottle is the, “Nasal Spray Pump w/ Safety Clip, Pfeiffer SAP #60548, which delivers a dose of 0.1 mL per squirt and has a diptubelength of 36.05 mm. It can be purchased from Pfeiffer of America ofPrinceton, N.J. Intranasal doses of a Y2 receptor-binding peptide suchas PYY can range from 0.1 μg/kg to about 1500 μg/kg. When administeredin as an intranasal spray, it is preferable that the particle size ofthe spray are between 10-100 μm (microns) in size, preferably 20-100 μmin size.

[0306] To promote weight loss, an intranasal dose of a Y2receptor-binding peptide PYY is administered at dose high enough topromote satiety but low enough so as not to induce any unwantedside-effects such as nausea. A preferred intranasal dose of a Y2receptor-binding peptide such as PYY(3-36) is about 3 μg-10 μg/kg weightof the patient, most preferably about 6 μg/kg weight of the patient. Ina standard dose a patient will receive 50 μg to 800 μg, more preferablyabout between 100 μg to 400 μg, most preferably 150 μg to about 200 μg.The a Y2 receptor-binding peptide such as PYY(3-36) is preferablyadministered at least ten minutes to one hour prior to eating to preventnausea but no more than about twelve to twenty-four hours prior toeating. The patient is dosed at least once a day preferably before everymeal until the patient has lost a desired amount of weight. The patientthen receives maintenance doses at least once a week preferably daily tomaintain the weight loss.”

[0307] As is shown by the data from the following examples, whenadministered intranasally to humans using the Y2 receptor-bindingpeptide formulation of the present invention, PYY(3-36) was found toreduce appetite. The examples also show that for the first timepost-prandial physiological levels of a PYY peptide could be reachedthrough an intranasal route of administration using the Y2receptor-binding peptide formulations of the present invention in whichPYY(3-36) was the Y2 receptor-binding peptide.

[0308] The following examples are provided by way of illustration, notlimitation.

EXAMPLE 1

[0309] An exemplary formulation for enhanced nasal mucosal delivery ofpeptide YY following the teachings of the instant specification wasprepared and evaluated as follows: TABLE 1 Peptide YY formulationcomposition Peptide YY₃₋₃₆ Formulations Per 100 ml Sample MucosalDelivery Enhancing Agent A 60 μg Phosphate-buffered saline (0.8%) pH 7.4(Control 1) B 60 μg Phosphate-buffered saline (0.8%) pH 5.0 (Control 2)C 60 μg L-Arginine (10% w/v) D 60 μg Poly-L-Arginine (0.5% w/v) E 60 μgGamma-Cyclodextrin (1% w/v) F 60 μg α-Cyclodextrin (5% w/v) G 60 μgMethyl-β-Cyclodextrin (3% w/v) H 60 μg n-Capric Acid Sodium (0.075% w/v)I 60 μg Chitosan (0.5% w/v) J 60 μg L-α-phosphatidilcholine didecanyl(3.5% w/v) K 60 μg S-Nitroso-N-Acetyl-Penicillamine (0.5% w/v) L 60 μgPalmotoyl-DL-Carnitine (0.02% w/v) M 60 μg Pluronic-127 (0.3% w/v) N 60μg Sodium Nitroprusside (0.3% w/v) O 60 μg Sodium Glycocholate (1% w/v)P 60 μg F1: Gelatin, DDPC, MBCD, EDTA F 1 L-α-phosphatidilcholinedidecanyl (0.5% w/v) Methyl β Cyclodextrin (3% w/v) EDTA (0.1% w/v, Inf.Conc. 0.5 M) Gelatin (0.5% w/v)

EXAMPLE 2

[0310] Nasal Mucosal Delivery—Permeation Kinetics and Cytotoxicity

[0311] 1. Organotypic Model

[0312] The following methods are generally useful for evaluating nasalmucosal delivery parameters, kinetics and side effects for peptide YYwithin the formulations and method of the invention, as well as fordetermining the efficacy and characteristics of the various intranasaldelivery-enhancing agents disclosed herein for combinatorial formulationor coordinate administration with peptide YY.

[0313] Permeation kinetics and cytotoxicity are also useful fordetermining the efficacy and characteristics of the various mucosaldelivery-enhancing agents disclosed herein for combinatorial formulationor coordinate administration with mucosal delivery-enhancing agents. Inone exemplary protocol, permeation kinetics and lack of unacceptablecytotoxicity are demonstrated for an intranasal delivery-enhancing agentas disclosed above in combination with a biologically active therapeuticagent, exemplified by peptide YY.

[0314] The EpiAirway system was developed by MatTek Corp (Ashland,Mass.) as a model of the pseudostratified epithelium lining therespiratory tract. The epithelial cells are grown on porousmembrane-bottomed cell culture inserts at an air-liquid interface, whichresults in differentiation of the cells to a highly polarizedmorphology. The apical surface is ciliated with a microvillousultrastructure and the epithelium produces mucus (the presence of mucinhas been confirmed by immunoblotting). The inserts have a diameter of0.875 cm, providing a surface area of 0.6 cm². The cells are plated ontothe inserts at the factory approximately three weeks before shipping.One “kit” consists of 24 units.

[0315] A. On arrival, the units are placed onto sterile supports in6-well microplates. Each well receives 5 mL of proprietary culturemedium. This DMEM-based medium is serum free but is supplemented withepidermal growth factor and other factors. The medium is always testedfor endogenous levels of any cytokine or growth factor, which is beingconsidered for intranasal delivery, but has been free of all cytokinesand factors studied to date except insulin. The 5 mL volume is justsufficient to provide contact to the bottoms of the units on theirstands, but the apical surface of the epithelium is allowed to remain indirect contact with air. Sterile tweezers are used in this step and inall subsequent steps involving transfer of units to liquid-containingwells to ensure that no air is trapped between the bottoms of the unitsand the medium.

[0316] B. The units in their plates are maintained at 37° C. in anincubator in an atmosphere of 5% CO₂ in air for 24 hours. At the end ofthis time the medium is replaced with fresh medium and the units arereturned to the incubator for another 24 hours.

[0317] 2. Experimental Protocol—Permeation Kinetics

[0318] A. A “kit” of 24 EpiAirway units can routinely be employed forevaluating five different formulations, each of which is applied toquadruplicate wells. Each well is employed for determination ofpermeation kinetics (4 time points), transepithelial resistance,mitochondrial reductase activity as measured by MTT reduction, andcytolysis as measured by release of LDH. An additional set of wells isemployed as controls, which are sham treated during determination ofpermeation kinetics, but are otherwise handled identically to the testsample-containing units for determinations of transepithelial resistanceand viability. The determinations on the controls are routinely alsomade on quadruplicate units, but occasionally we have employedtriplicate units for the controls and have dedicated the remaining fourunits in the kit to measurements of transepithelial resistance andviability on untreated units or we have frozen and thawed the units fordeterminations of total LDH levels to serve as a reference for 100%cytolysis.

[0319] B. In all experiments, the nasal mucosal delivery formulation tobe studied is applied to the apical surface of each unit in a volume of100 μL, which is sufficient to cover the entire apical surface. Anappropriate volume of the test formulation at the concentration appliedto the apical surface (no more than 100 μL is generally needed) is setaside for subsequent determination of concentration of the activematerial by ELISA or other designated assay.

[0320] C. The units are placed in 6 well plates without stands for theexperiment: each well contains 0.9 mL of medium which is sufficient tocontact the porous membrane bottom of the unit but does not generate anysignificant upward hydrostatic pressure on the unit.

[0321] D. To minimize potential sources of error and avoid any formationof concentration gradients, the units are transferred from one 0.9mL-containing well to another at each time point in the study. Thesetransfers are made at the following time points, based on a zero time atwhich the 100 μL volume of test material was applied to the apicalsurface: 15 minutes, 30 minutes, 60 minutes, and 120 minutes.

[0322] E. In between time points the units in their plates are kept inthe 37° C. incubator. Plates containing 0.9 mL medium per well are alsomaintained in the incubator so that minimal change in temperature occursduring the brief periods when the plates are removed and the units aretransferred from one well to another using sterile forceps.

[0323] F. At the completion of each time point, the medium is removedfrom the well from which each unit was transferred, and aliquotted intotwo tubes (one tube receives 700 μL and the other 200 μL) fordetermination of the concentration of permeated test material and, inthe event that the test material is cytotoxic, for release of thecytosolic enzyme, lactic dehydrogenase, from the epithelium. Thesesamples are kept in the refrigerator if the assays are to be conductedwithin 24 hours, or the samples are subaliquotted and kept frozen at−80° C. until thawed once for assays. Repeated freeze-thaw cycles are tobe avoided.

[0324] G. In order to minimize errors, all tubes, plates, and wells areprelabeled before initiating an experiment.

[0325] H. At the end of the 120 minute time point, the units aretransferred from the last of the 0.9 mL containing wells to 24-wellmicroplates, containing 0.3 mL medium per well. This volume is againsufficient to contact the bottoms of the units, but not to exert upwardhydrostatic pressure on the units. The units are returned to theincubator prior to measurement of transepithelial resistance.

[0326] 3. Experimental Protocol—Transepithelial Resistance

[0327] A. Respiratory airway epithelial cells form tight junctions invivo as well as in vitro, restricting the flow of solutes across thetissue. These junctions confer a transepithelial resistance of severalhundred ohms×cm² in excised airway tissues; in the MatTek EpiAirwayunits, the transepithelial resistance (TER) is claimed by themanufacturer to be routinely around 1000 ohms×cm². We have found thatthe TER of control EpiAirway units which have been sham-exposed duringthe sequence of steps in the permeation study is somewhat lower (700-800ohms×cm²), but, since permeation of small molecules is proportional tothe inverse of the TER, this value is still sufficiently high to providea major barrier to permeation. The porous membrane-bottomed unitswithout cells, conversely, provide only minimal transmembrane resistance(5-20 ohms×cm²).

[0328] B. Accurate determinations of TER require that the electrodes ofthe ohmmeter be positioned over a significant surface area above andbelow the membrane, and that the distance of the electrodes from themembrane be reproducibly controlled. The method for TER determinationrecommended by MatTek and employed for all experiments here employs an“EVOM”™ epithelial voltohmmeter and an “ENDOHM”™ tissue resistancemeasurement chamber from World Precision Instruments, Inc., Sarasota,Fla.

[0329] C. The chamber is initially filled with Dulbecco's phosphatebuffered saline (PBS) for at least 20 minutes prior to TERdeterminations in order to equilibrate the electrodes.

[0330] D. Determinations of TER are made with 1.5 mL of PBS in thechamber and 350 μL of PBS in the membrane-bottomed unit being measured.The top electrode is adjusted to a position just above the membrane of aunit containing no cells (but containing 350 μL of PBS) and then fixedto ensure reproducible positioning. The resistance of a cell-free unitis typically 5-20 ohms×cm² (“background resistance”).

[0331] E. Once the chamber is prepared and the background resistance isrecorded, units in a 24-well plate which had just been employed inpermeation determinations are removed from the incubator andindividually placed in the chamber for TER determinations.

[0332] F. Each unit is first transferred to a petri dish containing PBSto ensure that the membrane bottom is moistened. An aliquot of 350 μLPBS is added to the unit and then carefully aspirated into a labeledtube to rinse the apical surface. A second wash of 350 μL PBS is thenapplied to the unit and aspirated into the same collection tube.

[0333] G. The unit is gently blotted free of excess PBS on its exteriorsurface only before being placed into the chamber (containing a fresh1.5 mL aliquot of PBS). An aliquot of 350 μL PBS is added to the unitbefore the top electrode is placed on the chamber and the TER is read onthe EVOM meter.

[0334] H. After the TER of the unit is read in the ENDOHM chamber, theunit is removed, the PBS is aspirated and saved, and the unit isreturned with an air interface on the apical surface to a 24-well platecontaining 0.3 mL medium per well.

[0335] I. The units are read in the following sequence: all sham-treatedcontrols, followed by all formulation-treated samples, followed by asecond TER reading of each of the sham-treated controls. After all theTER determinations are complete, the units in the 24-well microplate arereturned to the incubator for determination of viability by MTTreduction.

[0336] 4. Experimental Protocol—Viability by MTT Reduction

[0337] MTT is a cell-permeable tetrazolium salt which is reduced bymitochondrial dehydrogenase activity to an insoluble colored formazan byviable cells with intact mitochondrial function or by nonmitochondrialNAD(P)H dehydrogenase activity from cells capable of generating arespiratory burst. Formation of formazan is a good indicator ofviability of epithelial cells since these cells do not generate asignificant respiratory burst. We have employed a MTT reagent kitprepared by MatTek Corp for their units in order to assess viability.

[0338] A. The MTT reagent is supplied as a concentrate and is dilutedinto a proprietary DMEM-based diluent on the day viability is to beassayed (typically the afternoon of the day in which permeation kineticsand TER were determined in the morning). Insoluble reagent is removed bya brief centrifugation before use. The final MTT concentration is 1mg/mL B. The final MTT solution is added to wells of a 24-wellmicroplate at a volume of 300 μL per well. As has been noted above, thisvolume is sufficient to contact the membranes of the EpiAirway units butimposes no significant positive hydrostatic pressure on the cells.

[0339] C. The units are removed from the 24-well plate in which theywere placed after TER measurements, and after removing any excess liquidfrom the exterior surface of the units, they are transferred to theplate containing MTT reagent. The units in the plate are then placed inan incubator at 37° C. in an atmosphere of 5% CO₂ in air for 3 hours.

[0340] D. At the end of the 3-hour incubation, the units containingviable cells will have turned visibly purple. The insoluble formazanmust be extracted from the cells in their units to quantitate the extentof MTT reduction. Extraction of the formazan is accomplished bytransferring the units to a 24-well microplate containing 2 mLextractant solution per well, after removing excess liquid from theexterior surface of the units as before. This volume is sufficient tocompletely cover both the membrane and the apical surface of the units.Extraction is allowed to proceed overnight at room temperature in alight-tight chamber. MTT extractants traditionally contain highconcentrations of detergent, and destroy the cells.

[0341] E. At the end of the extraction, the fluid from within each unitand the fluid in its surrounding well are combined and transferred to atube for subsequent aliquotting into a 96-well microplate (200 μLaliquots are optimal) and determination of absorbance at 570 nm on aVMax multiwell microplate spectrophotometer. To ensure that turbidityfrom debris coming from the extracted units does not contribute to theabsorbance, the absorbance at 650 nm is also determined for each well inthe VMax and is automatically subtracted from the absorbance at 570 nm.The “blank” for the determination of formazan absorbance is a 200 μLaliquot of extractant to which no unit had been exposed. This absorbancevalue is assumed to constitute zero viability.

[0342] F. Two units from each kit of 24 EpiAirway units are leftuntreated during determination of permeation kinetics and TER. Theseunits are employed as the positive control for 100% cell viability. Inall the studies we have conducted, there has been no statisticallysignificant difference in the viability of the cells in these untreatedunits vs cells in control units which had been sham treated forpermeation kinetics and on which TER determinations had been performed.The absorbance of all units treated with test formulations is assumed tobe linearly proportional to the percent viability of the cells in theunits at the time of the incubation with MTT. It should be noted thatthis assay is carried out typically no sooner than four hours afterintroduction of the test material to the apical surface, and subsequentto rinsing of the apical surface of the units during TER determination.

[0343] 5. Determination of Viability by LDH Release

[0344] While measurement of mitochondrial reductase activity by MTTreduction is a sensitive probe of cell viability, the assay necessarilydestroys the cells and therefore can be carried out only at the end ofeach study. When cells undergo necrotic lysis, their cytotosoliccontents are spilled into the surrounding medium, and cytosolic enzymessuch as lactic dehydrogenase (LDH) can be detected in this medium. Anassay for LDH in the medium can be performed on samples of mediumremoved at each time point of the two-hour determination of permeationkinetics. Thus, cytotoxic effects of formulations which do not developuntil significant time has passed can be detected as well as effects offormulations which induce cytolysis with the first few minutes ofexposure to airway epithelium.

[0345] A. The recommended LDH assay for evaluating cytolysis of theEpiAirway units is based on conversion of lactate to pyruvate withgeneration of NADH from NAD. The NADH is then reoxidized along withsimultaneous reduction of the tetrazolium salt INT, catalyzed by a crude“diaphorase” preparation. The formazan formed from reduction of INT issoluble, so that the entire assay for LDH activity can be carried out ina homogenous aqueous medium containing lactate, NAD, diaphorase, andINT.

[0346] B. The assay for LDH activity is carried out on 50 μL aliquotsfrom samples of “supernatant” medium surrounding an EpiAirway unit andcollected at each time point. These samples were either stored for nolonger than 24 h in the refrigerator or were thawed after being frozenwithin a few hours after collection. Each EpiAirway unit generatessamples of supernatant medium collected at 15 min, 30 min, 1 h, and 2 hafter application of the test material. The aliquots are all transferredto a 96 well microplate.

[0347] C. A 50 μL aliquot of medium which had not been exposed to a unitserves as a “blank” or negative control of 0% cytotoxicity. We havefound that the apparent level of “endogenous” LDH present after reactionof the assay reagent mixture with the unexposed medium is the samewithin experimental error as the apparent level of LDH released by allthe sham-treated control units over the entire time course of 2 hoursrequired to conduct a permeation kinetics study. Thus, withinexperimental error, these sham-treated units show no cytolysis of theepithelial cells over the time course of the permeation kineticsmeasurements.

[0348] D. To prepare a sample of supernatant medium reflecting the levelof LDH released after 100% of the cells in a unit have lysed, a unitwhich had not been subjected to any prior manipulations is added to awell of a 6-well microplate containing 0.9 mL of medium as in theprotocol for determination of permeation kinetics, the plate containingthe unit is frozen at −80° C., and the contents of the well are thenallowed to thaw. This freeze-thaw cycle effectively lyses the cells andreleases their cytosolic contents, including LDH, into the supernatantmedium. A 50 μL aliquot of the medium from the frozen and thawed cellsis added to the 96-well plate as a positive control reflecting 100%cytotoxicity.

[0349] E. To each well containing an aliquot of supernatant medium, a 50μL aliquot of the LDH assay reagent is added. The plate is thenincubated for 30 minutes in the dark.

[0350] F. The reactions are terminated by addition of a “stop” solutionof 1 M acetic acid, and within one hour of addition of the stopsolution, the absorbance of the plate is determined at 490 nm.

[0351] G. Computation of percent cytolysis is based on the assumption ofa linear relationship between absorbance and cytolysis, with theabsorbance obtained from the medium alone serving as a reference for 0%cytolysis and the absorbance obtained from the medium surrounding afrozen and thawed unit serving as a reference for 100% cytolysis.

[0352] 6. ELISA Determinations

[0353] The procedures for determining the concentrations of biologicallyactive agents as test materials for evaluating enhanced permeation ofactive agents in conjunction with coordinate administration of mucosaldelivery-enhancing agents or combinatorial formulation of the inventionare generally as described above and in accordance with known methodsand specific manufacturer instructions of ELISA kits employed for eachparticular assay. Permeation kinetics of the biologically active agentis generally determined by taking measurements at multiple time points(for example 15 min., 30 min., 60 min. and 120 min) after thebiologically active agent is contacted with the apical epithelial cellsurface (which may be simultaneous with, or subsequent to, exposure ofthe apical cell surface to the mucosal delivery-enhancing agent(s)).

[0354] The procedures for determining the concentrations of peptide YYneuropeptide Y, and pancreatic peptide in blood serum, central nervoussystem (CNS) tissues or fluids, cerebral spinal fluid (CSF), or othertissues or fluids of a mammalian subject may be determined byimmunologic assay for peptide YY neuropeptide Y, and pancreatic peptide.The procedures for determining the concentrations of peptide YYneuropeptide Y, and pancreatic peptide as test materials for evaluatingenhanced permeation of active agents in conjunction with coordinateadministration of mucosal delivery-enhancing agents or combinatorialformulation of the invention are generally as described above and inaccordance with known methods and specific manufacturer instructions forradioimmunoassay (RIA), enzyme immunoassay (EIA), and antibody reagentsfor immunohistochemistry or immunofluorescence for peptide YYneuropeptide Y, and pancreatic peptide. Bachem AG (King of Prussia,Pa.).

[0355] EpiAirway™ tissue membranes are cultured in phenol red andhydrocortisone free medium (MatTek Corp., Ashland, Mass.). The tissuemembranes are cultured at 37° C. for 48 hours to allow the tissues toequilibrate. Each tissue membrane is placed in an individual well of a6-well plate containing 0.9 mL of serum free medium. 100 μL of theformulation (test sample or control) is applied to the apical surface ofthe membrane. Triplicate or quadruplicate samples of each test sample(mucosal delivery-enhancing agent in combination with a biologicallyactive agent, peptide YY) and control (biologically active agent,peptide YY, alone) are evaluated in each assay. At each time point (15,30, 60 and 120 minutes) the tissue membranes are moved to new wellscontaining fresh medium. The underlying 0.9 mL medium samples isharvested at each time point and stored at 4° C. for use in ELISA andlactate dehydrogenase (LDH) assays.

[0356] The ELISA kits are typically two-step sandwich ELISAs: theimmunoreactive form of the agent being studied is first “captured” by anantibody immobilized on a 96-well microplate and after washing unboundmaterial out of the wells, a “detection” antibody is allowed to reactwith the bound immunoreactive agent. This detection antibody istypically conjugated to an enzyme (most often horseradish peroxidase)and the amount of enzyme bound to the plate in immune complexes is thenmeasured by assaying its activity with a chromogenic reagent. Inaddition to samples of supernatant medium collected at each of the timepoints in the permeation kinetics studies, appropriately diluted samplesof the formulation (i.e., containing the subject biologically activetest agent) that was applied to the apical surface of the units at thestart of the kinetics study are also assayed in the ELISA plate, alongwith a set of manufacturer-provided standards. Each supernatant mediumsample is generally assayed in duplicate wells by ELISA (it will berecalled that quadruplicate units are employed for each formulation in apermeation kinetics determination, generating a total of sixteen samplesof supernatant medium collected over all four time points).

[0357] A. It is not uncommon for the apparent concentrations of activetest agent in samples of supernatant medium or in diluted samples ofmaterial applied to the apical surface of the units to lie outside therange of concentrations of the standards after completion of an ELISA.No concentrations of material present in experimental samples aredetermined by extrapolation beyond the concentrations of the standards;rather, samples are rediluted appropriately to generate concentrationsof the test material which can be more accurately determined byinterpolation between the standards in a repeat ELISA.

[0358] B. The ELISA for a biologically active test agent, for example,peptide YY, is unique in its design and recommended protocol. Unlikemost kits, the ELISA employs two monoclonal antibodies, one for captureand another, directed towards a nonoverlapping determinant for thebiologically active test agent, e.g., peptide YY, as the detectionantibody (this antibody is conjugated to horseradish peroxidase). Aslong as concentrations of peptide YY that lie below the upper limit ofthe assay are present in experimental samples, the assay protocol can beemployed as per the manufacturer's instructions, which allow forincubation of the samples on the ELISA plate with both antibodiespresent simultaneously. When the peptide YY levels in a sample aresignificantly higher than this upper limit, the levels of immunoreactivepeptide YY may exceed the amounts of the antibodies in the incubationmixture, and some peptide YY which has no detection antibody bound willbe captured on the plate, while some peptide YY which has detectionantibody bound may not be captured. This leads to seriousunderestimation of the peptide YY levels in the sample (it will appearthat the peptide YY levels in such a sample lie significantly below theupper limit of the assay). To eliminate this possibility, the assayprotocol has been modified:

[0359] B.1. The diluted samples are first incubated on the ELISA platecontaining the immobilized capture antibody for one hour in the absenceof any detection antibody. After the one hour incubation, the wells arewashed free of unbound material.

[0360] B.2. The detection antibody is incubated with the plate for onehour to permit formation of immune complexes with all captured antigen.The concentration of detection antibody is sufficient to react with themaximum level of peptide YY which has been bound by the captureantibody. The plate is then washed again to remove any unbound detectionantibody.

[0361] B.3. The peroxidase substrate is added to the plate and incubatedfor fifteen minutes to allow color development to take place.

[0362] B.4. The “stop” solution is added to the plate, and theabsorbance is read at 450 nm as well as 490 nm in the VMax microplatespectrophotometer. The absorbance of the colored product at 490 nm ismuch lower than that at 450 nm, but the absorbance at each wavelength isstill proportional to concentration of product. The two readings ensurethat the absorbance is linearly related to the amount of bound peptideYY over the working range of the VMax instrument (we routinely restrictthe range from 0 to 2.5 OD, although the instrument is reported to beaccurate over a range from 0 to 3.0 OD). The amount of peptide YY in thesamples is determined by interpolation between the OD values obtainedfor the different standards included in the ELISA. Samples with ODreadings outside the range obtained for the standards are rediluted andrun in a repeat ELISA.

RESULTS

[0363] Measurement of transepithelial resistance by TER Assay: After thefinal assay time points, membranes were placed in individual wells of a24 well culture plate in 0.3 mL of clean medium and the trans epithelialelectrical resistance (TER) was measured using the EVOM EpithelialVoltohmmeter and an Endohm chamber (World Precision Instruments,Sarasota, Fla.). The top electrode was adjusted to be close to, but notin contact with, the top surface of the membrane. Tissues were removed,one at a time, from their respective wells and basal surfaces wererinsed by dipping in clean PBS. Apical surfaces were gently rinsed twicewith PBS. The tissue unit was placed in the Endohm chamber, 250 μL ofPBS added to the insert, the top electrode replaced and the resistancemeasured and recorded. Following measurement, the PBS was decanted andthe tissue insert was returned to the culture plate. All TER values arereported as a function of the surface area of the tissue.

[0364] The final numbers were calculated as:

TER of cell membrane=(Resistance (R) of Insert with membrane−R of blankInsert)×Area of membrane (0.6 cm²).

[0365] Exemplary peptide YY formulation, Formulation P, showed thegreatest decrease in cell membrane resistance. (Table 2). The resultsindicate that the exemplary formulation (e.g., Formulation P) reducesthe resistance of the membrane to less than 1% of the control at theconcentrations tested. The values shown are the average of threereplicates of each formulation. Formulations A and B are controlsprepared by reconstituting peptide YY (Bachem AG, King of Prussia, Pa.)containing 60 μg peptide Y3-36 in 100 ml of phosphate buffered saline(PBS) at pH 7.4 or 5.0. Peptide YY without mucosal delivery enhancersdid not decrease the resistance.

[0366] The results indicate that an exemplary formulation for enhancedintranasal delivery of peptide YY (e.g., Formulation P) decreases cellmembrane resistance and significantly increases mucosal epithelial cellspermeability. The exemplary formulations will enhance intranasaldelivery of peptide YY to the blood serum or to the central nervoussystem tissue or fluid. The results indicate that these exemplaryformulations when contacted with a mucosal epithelium yield significantincreases in mucosal epithelial cell permeability to peptide YY. TABLE 2Influence of Pharmaceutical Formulations Comprising Peptide YY andIntranasal Delivery-Enhancing Agents on Transepithelial Resistance (TER)of EpiAirway Cell Membrane Mucosal Delivery Enhancing Formulation Agent% TER A PBS pH 7.4 (Control 1) 100 B PBS pH 5.0 (Control 2) 100 CL-Arginine (10% w/v) 47.88 D Poly-L-Arginine (0.5% w/v) 3.96 EGamma-Cyclodextrin 91.67 (1% w/v) F Alpha-Cyclodextrin (5% w/v) 88.91 GMethyl-β-Cyclodextrin 97.51 (3% w/v) H n-Capric Acid Sodium 47.72(0.075% w/v) I Chitosan (0.5% w/v) 4.77 J L-α-phosphatidilcholine 0.49didecanyl (3.5% w/v) K S-Nitroso-N-Acetyl- 44.35 Penicillamine (0.5%w/v) L Palmotoyl-DL-Carnitine 1.76 (0.02% w/v) M Pluronic-127 (0.3% w/v)97.57 N Sodium Nitroprusside 92.41 (0.3% w/v) O Sodium Glycocholate14.25 (1% w/v) P F1: Gelatin, DDPC, MBCD, 0.65 EDTA

[0367] Permeation kinetics as measured by ELISA Assay: The effect ofpharmaceutical formulations of the present invention comprising peptideYY and intranasal delivery-enhancing agents on the permeation of peptideYY across the EpiAirway Cell Membrane (mucosal epithelial cell layer) ismeasured as described above. The results are shown in Table 3.Permeation of peptide YY across the EpiAirway™ Cell Membrane is measuredby ELISA assay.

[0368] For the exemplary intranasal formulations (e.g., Formulation P)of the present invention, the greatest increase in peptide YY permeationoccurred in Formulation P as shown in Table 3. The procedure uses anELISA assay to determine the concentration of biologically activepeptide YY that has permeated the epithelial cells into the surroundingmedium over multiple time points. The results show increased permeationof peptide YY in Formulation P compared to Formulation A or B (peptideYY control formulation; 60 μg peptide YY₃₋₃₆ in 100 ml of phosphatebuffered saline (PBS) at pH 7.4 or 5.0; Bachem AG, King of Prussia,Pa.). On average the cumulative increase in permeation at 120 minutesusing Formulation P exemplary intranasal formulation is about 1195 foldgreater than Formulations A or B controls. TABLE 3 Influence ofPharmaceutical Formulations Comprising Peptide YY and IntranasalDelivery-Enhancing Agents on Permeation of Peptide YY through EpiAirwayCell Membrane by ELISA Assay. Formulation % Permeation at Time Points(min) Total % Fold Increase Peptide YY₃₋₃₆ (60 μg/100 ml) 0 15 30 60 120Permeation in Permeability A PBS pH 7.4 (Control 1) 0 0.00171 0.000960.00451 0.00327 0.01 1 B PBS pH 5.0 (Control 2) 0 0.00093 0.000480.00042 0.00367 0.01 1 C L-Arginine (10% w/v) 0 0.00119 0.00277 0.006850.00566 0.02 2 D Poly-L-Arginine (0.5% w/v) 0 0.00324 0.01587 0.103950.49656 0.62 62 E Gamma-Cyclodextrin (1% w/v) 0 0.00017 0.00042 0.000280.0035 0 1 F α-Cyclodextrin (5% w/v) 0 0.00031 0.000745 0.00147 0.00310.01 1 G Methyl-β-Cyclodextrin (3% w/v) 0 0.00028 0.00038 0.000590.01028 0.01 1 H n-Capric Acid Sodium (0.075% w/v) 0 0.0004 0.001310.00448 0.00821 0.01 1 I Chitosan (0.5% w/v) 0 0.00086 0.01098 0.097490.82126 0.93 93 J L-α-phosphatidilcholine didecanyl (3.5% w/v) 0 0.009340.02 0.08507 1.9642 2.08 208 K S-Nitroso-N-Acetyl-Penicillamine (0.5%w/v) 0 0.00074 0.0032 0.0688 0.90432 0.98 98 L Palmotoyl-DL-Carnitine(0.02% w/v) 0 0.00378 0.03422 0.15141 1.31011 1.5 150 M Pluronic-127(0.3% w/v) 0 0.00025 0.00027 0.00066 0.00395 0.01 1 N SodiumNitroprusside (0.3% w/v) 0 0.00171 0.00114 0.00079 0.05492 0.05 5 OSodium Glycocholate (1% w/v) 0 0.00325 0.00313 0.09023 0.70214 0.8 80 PF1 Gelatin, DDPC, MBCD, EDTA 0 0.05864 1.3972 2.9799 7.519 11.95 1195

[0369] MTT Assay: The MTT assays were performed using MTT-100, MatTekkits. 300 mL of the MTT solution was added into each well. Tissueinserts were gently rinsed with clean PBS and placed in the MTTsolution. The samples were incubated at 37° C. for 3 hours. Afterincubation the cell culture inserts were then immersed with 2.0 mL ofthe extractant solution per well to completely cover each insert. Theextraction plate was covered and sealed to reduce evaporation.Extraction proceeds overnight at RT in the dark. After the extractionperiod was complete, the extractant solution was mixed and pipetted intoa 96-well microtiter plate. Triplicates of each sample were loaded, aswell as extractant blanks. The optical density of the samples was thenmeasured at 550 nm on a plate reader (Molecular Devices).

[0370] The MTT assay on an exemplary formulation for enhanced nasalmucosal delivery of peptide YY following the teachings of the instantspecification (e.g., Formulation P) compared to control formulation(Formulations A or B) are shown in Table 4. The results for formulationscomprising peptide YY and one or more intransal delivery enhancingagents, for example, Formulation P (experiment performed in threereplicates) indicate that there is minimal toxic effect of thisexemplary embodiment on viability of the mucosal epithelial tissue.TABLE 4 Influence of Pharmaceutical Formulations Comprising Peptide YYand Intranasal Delivery-Enhancing Agents on the Viability of EpiAirwayCell Membrane as shown by % MTT Formulations Treatment % MTT A PBS pH.4(Control 1) 100 B PBS pH 5.0 (Control 2) 100 C L-Arginine (10% w/v)91.54 D Poly-L-Arginine (0.5% w/v) 79.39 E Gamma-Cyclodextrin (1% w/v)100 F α-Cyclodextrin (5% w/v) 96.63 G Methyl-β-Cyclodextrin (3% w/v) 100H n-Capric Acid Sodium 100 (0.075% w/v) I Chitosan (0.5% w/v) 100 JL-α-phosphatidilcholine 94.25 didecanyl (3.5% w/v) K S-Nitroso-N-Acetyl-97.64 Penicillamine (0.5% w/v) L Palmotoyl-DL-Carnitine 91.77 (0.02%w/v) M Pluronic-127 (0.3% w/v) 100 N Sodium Nitroprusside 100 (0.3% w/v)O Sodium Glycocholate (1% w/v) 100 P F1: Gelatin, DDPC, MBCD, 88.75 EDTA

[0371] LDH Assay: The LDH assay on an exemplary formulation for enhancednasal mucosal delivery of peptide YY following the teachings of theinstant specification (e.g., Formulation P) are shown in Table 5. Theresults for three replicates of Formulation P indicate that there isminimal toxic effect of this exemplary embodiment on viability of themucosal epithelial tissue. TABLE 5 Influence of PharmaceuticalFormulations Comprising Peptide YY and Intranasal Delivery-EnhancingAgents on the Viability of EpiAirway Cell Membrane as shown by % DeadCells (LDH Assay) Formulations Treatment % dead cells A PBS pH .4(Control 1) 1.0 B PBS pH 5.0 (Control 2) 1.1 C L-Arginine (10% w/v) 0.8D Poly-L-Arginine (0.5% w/v) 1.4 E Gamma-Cyclodextrin (1% w/v) 0.8 Fα-Cyclodextrin (5% w/v) 0.7 G Methyl-β-Cyclodextrin (3% w/v) 0.8 Hn-Capric Acid Sodium 1.3 (0.075% w/v) I Chitosan (0.5% w/v) 0.7 JL-α-phosphatidilcholine 1.2 didecanyl (3.5% w/v) K S-Nitroso-N-Acetyl-0.7 Penicillamine (0.5% w/v) L Palmotoyl-DL-Carnitine 0.8 (0.02% w/v) MPluronic-127 (0.3% w/v) 1.0 N Sodium Nitroprusside 0.6 (0.3% w/v) OSodium Glycocholate (1% w/v) 0.8 P F1: Gelatin, DDPC, MBCD, 2.0 EDTA

EXAMPLE 3 Formulation P (Peptide YY) of the Present Invention InCombination With Triamcinolone Acetonide Corticosteroid Improves CellViability

[0372] The present example provides an in vitro study to determine thepermeability and reduction in epithelial mucosal inflammation of anintranasally administered peptide YY, for example, human peptide YY, incombination with a steroid composition, for example, triamcinoloneacetonide, and further in combination with one or more intranasaldelivery-enhancing agents. The study involves determination ofepithelial cell permeability by TER assay and reduction in epithelialmucosal inflammation as measured by cell viability in an MTT assay byapplication of an embodiment comprising peptide YY and triamcinoloneacetonide.

[0373] Formulation P (see Table 1 above) is combined in a formulationwith triamcinolone acetonide at a dosage of 0.5, 2.0, 5.0, or 50 μg.Normal dose of triamcinolone acetonide, (Nasacort®, AventisPharmaceuticals) for seasonal allergic rhinitis, is 55 μg per spray.Formulation P in combination with triamcinolone acetonide corticosteroidimproves cell viability as measured by the MTT assay, while maintainingepithelial cell permeability as measured by TER and ELISA assays.

[0374] According to the methods and formulations of the invention,measurement of permeability of Formulation P in the presence or absenceof triamcinolone acetonide is performed by transepithelial electricalresistance (TER) assays in an EpiAirway™ cell membrane. TER assays ofFormulation P plus triamcinolone acetonide at a concentration of 0.5,2.0, 5.0, or 50 μg per spray indicate that peptide YY permeability didnot decrease and was equal to permeability of Formulation P alone.Formulation P plus triamcinolone acetonide at a triamcinolone acetonideconcentration between 0 and 50 μg per spray is typically, at least10-fold to 100-fold greater than permeability of Formulations A or B(peptide YY control).

[0375] According to the methods and formulations of the invention,measurement of permeability of Formulation P in the presence or absenceof triamcinolone acetonide is performed by ELISA assay in an EpiAirway™cell membrane. Similar to the TER assay above, ELISA assay ofFormulation P plus triamcinolone acetonide at a concentration of 0.5,2.0, 5.0, or 50 μg per spray indicate that peptide YY permeability didnot decrease and was equal to permeability of Formulation P alone.Formulation P plus triamcinolone acetonide at a triamcinolone acetonideconcentration between 0 and 50 μg per spray is typically greater thanpermeability of Formulations A or B (peptide YY control).

[0376] According to the methods and formulations of the invention, MTTassay measured cell viability of Formulation P in the presence orabsence of triamcinolone acetonide. Typically, addition of triamcinoloneacetonide (at a concentration of 0.5, 2.0, 5.0, or 50 μg per spray) toFormulation P improves cell viability compared to Formulation P in theabsence of triamcinolone acetonide.

[0377] Addition of triamcinolone acetonide to Formulation P increasescell viability and maintains epithelial permeability as measured by TERassay comparable to Formulation P in the absence of triamcinoloneacetonide.

[0378] Reduction in epithelial mucosal inflammation of an intranasallyadministered peptide YY is accomplished with an intranasal formulationof peptide YY in combination with one or more steroid or corticosteroidcompound(s) typically high potency compounds or formulations, but alsoin certain cases medium potency, or low potency compounds orformulations. Overall potency (equivalent dosages) of high, medium, andlow potency steroids are given. Typically, an intranasal formulation ofpeptide YY in combination with a high potency steroid compositionincludes, but is not limited to, betamethasone (0.6 to 0.75 mg dosage),or dexamethasone (0.75 mg dosage). In an alternative formulation, anintranasal formulation of peptide YY in combination with a mediumpotency steroid composition includes, but is not limited to,methylprednisolone (4 mg dosage), triamcinolone (4 mg dosage), orprednisolone (5 mg dosage). In a further alternative formulation, anintranasal formulation of peptide YY in combination with a low potencysteroid composition includes, but is not limited to hydrocortisone (20mg dosage) or cortisone (25 mg dosage).

EXAMPLE 4 Preparation of a PYY Formulation Free of a Stabilizer that isa Protein

[0379] A PYY formulation suitable for intranasal administration of PYY,which was substantially free of a stabilizer that is a protein wasprepared having the formulation listed below.

[0380] 1. About ¾ of the water was added to a beaker and stirred with astir bar on a stir plate and the sodium citrate was added until it wascompletely dissolved.

[0381] 2. The EDTA was then added and stirred until it was completelydissolved.

[0382] 3. The citric acid was then added and stirred until it wascompletely dissolved.

[0383] 4. The methyl-β-cyclodextrin was added and stirred until it wascompletely dissolved.

[0384] 5. The DDPC was then added and stirred until it was completelydissolved.

[0385] 6. The lactose was then added and stirred until it was completelydissolved.

[0386] 7. The sorbitol was then added and stirred until it wascompletely dissolved.

[0387] 8. The chlorobutanol was then added and stirred until it wascompletely dissolved.

[0388] 9. The PYY 3-36 was added and stirred gently until it dissolved.

[0389] 10. 11 Check the pH to make sure it is 5.0±0.25. Add dilute HClor dilute NaOH to adjust the pH.

[0390] 11. Add water to final volume. TABLE 6 Reagent Grade Vendor mg/mL% Cholorbutanol, anhydrous NF Spectrum 5.0 0.50 Methyl-β-CyclodextrinSigma 45 4.5 L-α-Phospharidycholine Sigma 1 0.1 Didecanoyl EdetateDisodium USP Dow Chemicals 1 0.1 Sodium Citrate, Dihydrate USP Spectrum1.62 0.162 Citric Acid, Anhydrous USP Sigma 0.86 0.086 α-Lactosemonohydrate Sigma 9 0.9 Sorbitol Sigma 18.2 1.82 PYY 3-36 GMP Bachem 10.1 Purified Water

EXAMPLE 5

[0391] A second formulation was prepared as above, except theconcentration of PYY 3-36 was 15 mg/mL as shown below in Table 7. TABLE7 Reagent Grade Vendor mg/ml % Cholorbutanol, anhydrous NF Spectrum 5.00.50 Methyl-β-Cyclodextrin Sigma 45 4.5 L-α-Phospharidycholine Sigma 10.1 Didecanoyl Edetate Disodium USP Dow Chemicals 1 0.1 Sodium Citrate,Dihydrate USP Spectrum 1.62 0.162 Citric Acid, Anhydrous USP Sigma 0.860.086 α-Lactose monohydrate Sigma 9 0.9 Sorbitol Sigma 18.2 1.82 PYY3-36 GMP Bachem 15 0.1 Purified Water

EXAMPLE 6 Determination of Optimal pH of PYY

[0392] Determination of PYY₃₋₃₆ stability v's pH at 40° C. for 5 days

[0393] A. Protocol for Formulafing PYY (3-36)/pH Stability Study Samples

[0394] Osmolarity: Target 250 mM

[0395] Using a Citrate/Sodium citrate, tri-basic buffer, 10 mM$\begin{matrix}{{Osmolarity} = {{{no}.\quad {particles}} \times {molarity}}} & \quad \\{\quad {= {{( {1 + 4} ) \times 10\quad {mM}} = {50\quad {mM}}}}} & \quad\end{matrix}$

[0396] Therefore bring osmolarity to 250 mM with 100 mM NaCl (2particles)

[0397] B. Made Up Stability Samples As Follows (3500 μl)

[0398] Final Concentration

[0399] Citrate buffer 1400 μL 25 mM (of required final pH) 10 mM

[0400] PYY 700 μl 1.5 mg/mL 300 μg/ml

[0401] Chlorobutanol 350 μl 2.5%, 0.25%

[0402] NaCl, 350 μl 1.0M, 100 mM

[0403] Check pH and adjust if required

[0404] Q.S. to 3500 μL with water

[0405] Procedure:

[0406] 120 μl sample in 200 μl sialanized inserts in autosampler vials

[0407] 3 pulls/time point

[0408] Samples incubated at 40° C. for 5 days

[0409] C. Comparison of Target and Actual Final pH of Stability MixturesTarget pH Actual pH 3.0 2.99 3.5 3.47 4.0 3.90 4.5 4.42 5.0 4.90 7.07.38

[0410] D. HPLC Procedure Column: Waters C18 Bondapak 10 μm 4.6 × 300 mmHPLC system: Waters Alliance 2690 Detector: Waters 2487 Dual wavelengthat 220 nm Flow rate:  1 ml/mim Injection volume: 30 μL

[0411] Mobile Phases:

[0412] Buffer A: 0.1% TFA, 1% acetonitrile in water

[0413] Buffer B: 0.11% TFA in acetonitrile Gradient: Time (mins) % A % B0 75 25 17 42 58 19 75 25 28 75 25

[0414] E. Results and Conclusions:

[0415] Results indicate that under the particular conditions used inthis study, that the optimal pH for stability is 4.90. There is anincrease in stability from 76 to 87% with increasing pH from 2.99 to4.90.

[0416] At higher pH, i.e. 7.38 there is a large drop in the stability ofPYY(3-36) with only 15% of time zero remaining.

EXAMPLE 7 Intranasal Formulation Development

[0417] Peptides and proteins are relatively fragile molecules comparedto low-molecular-weight therapeutics. The objective of the formulationdevelopment phase was to identify a candidate formulation suitable forintranasal delivery. In order to achieve this goal, numerous candidateswere tested in order to identify a formulation with acceptable drugstability, delivery across the nasal mucosa, toxicity and preservativeeffectiveness.

[0418] Initially, the effect of pH was examined. FIG. 1 shows thestability of PYY 3-36 at high temperature (40° C.) at various pHs from3.0 to 7.4. At physiological pH, there was substantial loss of drug atelevated temperature. Best stability was achieved at about pH 5.0. ThispH was chosen for further formulation optimization.

[0419] To further optimize stability, various stabilizing agents weretested for their ability to facilitate passage of drug across the nasalmucosa. The enhancers tested were chosen based on their ability to opentight junctions with limited cellular toxicity. To accomplish this, aprimary human epithelial cell model (EpiAirway, MatTek, Inc., AshlandMass.) was employed. This cell line forms a pseudo-stratified columnarepithelial cell layer with tight junctions similar to the respiratoryepithelium found in the nose. Drug formulations were placed on theapical side of the tissue layer, and drug quantitation carried out forthe basal media. The extent of tight junction opening was measured bydecrease in the transepithelial electrical resistance (TEER). Cellviability and cytotoxicity were monitored by MTT and LDH assays,respectively. Data from a representative screening experiment aredepicted in FIGS. 2-5.

[0420]FIG. 2 shows the data for TEER. In some cases there was little orno decrease in TEER compared to the control, indicating tight junctionswhich remain closed. In other cases there was a substantial drop in TEERindicating tight junction opening. The results demonstrate that the invitro cell model is capable of discriminating the ability of differentformulations to open the tight junctions.

[0421] In the candidate formulations tested the cell viabilities (FIG.3; MTT) were good and cyctotoxicities (FIG. 4; LDH) were low.

[0422] In total, over 200 different formulations were tested, reflectingthe high-throughput nature of the in vitro screening model. Using allthe available data, a multivariate analysis was conducted to elucidatethe effect each formulation component exerted on each of the 7 outputvariables (drug permeability, osmolality, stability at refrigerated andaccelerated conditions, TEER, and MTT and LDH assays). The multivariateanalysis consisted of an initial analysis of each formulation componentfor some level of correlation with output parameters (p<0.1). With thesubset identified, either a linear regression or stepwise logisticselection model was used. The results suggest that one excipientcorrelated to osmolality and toxicity (r²=0.91 and 0.27, respectively),two correlated to PYY3-36 permeation (r²=0.44), three affected stability(r²=0.24), and five impacted paracellular resistance (r²=0.55). The bestformulations determined by this process increased at least 30-75 foldthe PYY 3-36 transport compared to simple buffer solutions.

[0423] Based on these analyses, an optimized PYY₃₋₃₆ formulation wasselected for further development. This optimized formulation containedtwo stabilizers, two permeation enhancers, one chelating agent, and onepreservative in a sodium acetate buffer, pH 5.0. This formulation passedthe USP Preservative Effectiveness Test. The synergistic contributionsof the various components on drug permeation is presented in FIG. 5.Compared to simple buffer formulations at the same osmolality, theoptimized formulation exhibits more than 100-fold increased drugpermeation.

[0424] Finally, pre-clinical and clinical batches of the optimizedformulation were prepared and placed on stability at 5° C. and 25° C. inthe final product packaging. Preliminary data, depicted in Table 8reveal that storage for up to two months at either 5° C. or 25° C.results in 90% or better peptide retention. TABLE 8 5° C. 25° C. Timepoint % PYY % PYY   0 100.0 100.0   3 days 99.8 100.1   7 days 102.698.4  10 days 103.3 101.7   2 weeks 101.4 97.9   3 weeks 100.3 95.4   1month 100.5 96.3 1.5 months 100.1 90.6   2 months 99.8 92.3

[0425] In summary, our process of formulation development has produced aPYY₃₋₃₆ formulation with suitable drug stability, delivery across thenasal mucosa, toxicity and preservative effectiveness, which enablesdelivery of a 4 kD peptide.

[0426] Preclinical Studies

[0427] To date, a series of six preclinical studies in rats, rabbits,and dogs have been completed. Plasma PYY3-36 levels in all species weredetermined by a validated proprietary radioimmunoassay method.

[0428] Bioavailability (the molar fraction of drug identified in plasmadivided by the amount administered nasally) in rats was determined to beapproximately 6%, and in rabbits is approximately 8%. These values mayunderstate the true bioavailability, as any peptide degradation inplasma before sampling, or degradation after sampling despite thepresence of a proteinase inhibitor, will decrease the measuredbioavailability.

[0429] Nasal toxicity has been evaluated in rat and rabbit models for upto 14 consecutive days at doses 50× the expected human clinical dose ona mg/kg basis. There were no microscopic or gross pathological findingsrelated to the test article. There were no clinical observations.

[0430] Systemic toxicity following intravenous administration wasevaluated in rat and rabbit models. At IV doses up to approx 160× theexpected human dose (400 ug/kg in the rat and 205 ugikg in the rabbit)there were no test article related microscopic or macroscopic findings.

[0431] Cardiovascular toxicity was assessed in the anesthetized dogmodel in a dose ranging study design. The highest dosage, an infusion ofPYY₃₋₃₆ up to 24 ug/kg over 60 minutes corresponded to 33× the expectedhuman dose on a body surface area basis. The resultant plasma levels, 30ng/mL, was approximately 380× the basal canine plasma level. At thisplasma level, there was no effect on arterial blood pressure, femoralblood flow, or QTc and only minor changes in heart rate (increase from123 to 148 bpm mean) and respiratory rate (decrease from 54 to 36) werenoted.

[0432] Pharmacokinetic data was collected in these preclinical studies.From one study in rats, the plasma levels following intranasaladministration at various doses are shown in FIGS. 6, 7 and 8. FIG. 6shows PYY3-36 is seen in the plasma within 5 minutes, peak plasmaconcentrations (Tmax) are reached in 10-15 minutes, and the terminalelimination half life is approx 15 minutes. Both C_(max) and AUC_(0-t)are linear with respect to intranasal dose.

[0433] Clinical Studies

[0434] A dose ranging clinical trial has been initiated with the goal ofestablishing safety, PK, and bioavailability of the intranasalformulation of PYY₃₋₃₆. To date, patients have been enrolled in thefirst two of five dose cohorts. One patient reported a taste in the backof his throat; there have been no other adverse events to date.

CONCLUSION

[0435] Formulation, preclinical, and initial clinical work have begun onan intranasal formulation of PYY₃₋₃₆. The approach to formulation hasresulted in a more than one hundred fold increase in transmembranepermeability of this 4 kD peptide with no increase in cellular toxicity.Preclinical studies have demonstrated a considerable safety margin fornasal, cardiovascular, and systemic toxicity for PYY₃₋₃₆. On the basison the ongoing dose ranging clinical studies, chronic administrationweight loss studies are planned.

EXAMPLE 8a Clinical PROTOCOL Nasal Absorption of Intranasal PeptideYY₃₋₃₆ (PYY₃₋₃₆) In Healthy Human Subjects

[0436] Object of the Present Study:

[0437] The object of the present study was to evaluate the absorption ofintranasally administered PYY3-36 into the blood stream from the nose.This was a phase I, in clinic, single dose, doses escalation studyinvolving fasted, normal, healthy male and female volunteers. Ascendingdoses of intranasal PYY3-36 were evaluated between 20 μg to 200 μg toevaluate safety, nasal tolerance and absorption of PYY3-36. Assessmentof appetite sensation in each individual was also evaluated.

[0438] PYY₃₋₃₆ was administered to 15 healthy humans divided into 5Groups of 3 individuals each.

[0439] Group I.

[0440] The first group was administered by an intranasal spray 20 μg ofPYY₃₋₃₆ in a 0.1 ml solution.

[0441] Group II

[0442] The second group received intranasally 50 μg of PYY₃₋₃₆ in a 0.1ml solution.

[0443] Group III

[0444] The third group received intranasally 100 μg of PYY₃₋₃₆ in a 0.1ml solution.

[0445] Group IV

[0446] The fourth group received intranasally 150 μg of PYY₃₋₃₆ in a 0.1ml solution.

[0447] Group V

[0448] The fifth group received intranasally 200 μg of PYY₃₋₃₆ in a 0.1ml solution.

[0449] Blood samples were taken collected and the plasma concentrationsof PYY were determined at 0 (i.e., pre-dose), 5, 7.5, 10, 15, 20, 30,45, 60 minutes post-dose. The subjects were then fed and a blood sampletaken and the concentration of PYY was determined 30 minutespostprandial. Plasma concentrations of PYY₃₋₃₆ were determined using avalidated analytical procedure.

[0450] For each subject, the following PK parameters were calculated,whenever possible, based on the plasma concentrations of PYY₃₋₃₆,according to the model independent approach:

[0451] C_(max) Maximum observed concentration.

[0452] t_(max) Time to maximum concentration.

[0453] AUC_(0-t) Area under the concentration-time curve from time 0 tothe time of last measurable concentration, calculated by the lineartrapezoidal rule.

[0454] The following parameters were calculated when the data permitsaccurate estimation of these parameters:

[0455] AUC_(0-∞) Area under the concentration-time curve extrapolated toinfinity, calculated using the formula:${AUC}_{0\text{-}\infty} = {{AUC}_{0\text{-}t} + \frac{C_{t}}{K_{e}}}$

[0456] where C_(t) is the last measurable concentration and K_(e) is theapparent terminal phase rate constant.

[0457] K_(e) Apparent terminal phase rate constant, where K_(e) is themagnitude of the slope of the linear regression of the log concentrationversus time profile during the terminal phase.

[0458] t_(1/2) Apparent terminal phase half-life (whenever possible),where t_(1/2)=(In2)/K_(e).

[0459] PK calculations were performed using commercial software such asWinNonlin (Pharsight Corporation, Version 3.3, or higher). The resultsare shown in the graphs below.

DISCUSSION AND CONCLUSION

[0460] Background:

[0461] Each dosing group included three subjects who were dosedintranasally once with a formulation of this invention that contained aspecified dose of synthetic, pyrogen-free human PYY₃₋₃₆. Five dosinggroups were organized, with escalating doses of PYY₃₋₃₆ in theformulation. Blood samples were drawn at specified intervals into bloodcollection tubes that contained lithium heparin (to inhibit coagulation)and aprotinin (to preserve PYY ₃₋₃₆)-Plasma from each blood sample wascollected by centrifugation and stored in frozen aliquots. One frozenaliquot of each blood sample was shipped to Nastech Analytical Servicesand arrived frozen. Each sample was stored frozen until assayed for PYYconcentration by radioimmunoassay (RIA).

[0462] Observations:

[0463] Group 1: This group of subjects was dosed with 20 micrograms ofPYY₃₋₃₆. Plasma PYY concentrations for the subjects varied from aminimum of “less than 20 pg/ml” (below the lower limit of quantitationof the radioimmunoassay) to a maximum of 159 pg/ml. The trends ofconcentrations observed are not consistent with significant absorptionof drug into the blood of the subjects studied.

[0464] Group 2: This group of subjects was dosed with 50 micrograms ofPYY₃₋₃₆. Plasma PYY concentrations for the subjects varied from aminimum of 50 pg/ml to a maximum of 255 pg/ml. The trends ofconcentrations observed are consistent with significant absorption ofdrug into the blood of the subjects studied.

[0465] Group 3: This group of subjects was dosed with 100 micrograms ofPYY₃₋₃₆. Plasma PYY concentrations for the subjects varied from aminimum of 87 pg/ml to a maximum of 785 pg/ml. The trends ofconcentrations observed are consistent with significant absorption ofdrug into the blood of the subjects studied.

[0466] Group 4: This group of subjects was dosed with 150 micrograms ofPYY₃₋₃₆. Plasma PYY concentrations for the subjects varied from aminimum of 45 pg/ml to a maximum of 2022 pg/ml. The trends ofconcentrations observed are consistent with significant absorption ofdrug into the blood of the subjects studied.

[0467] Group 5: This group of subjects was dosed with 200 micrograms ofPYY₃₋₃₆ Plasma PYY concentrations for the subjects varied from a minimumof 48 pg/ml to a maximum of 1279 pg/ml. The trends of concentrationsobserved are consistent with significant absorption of drug into theblood of the subjects studied.

[0468] These results are consistent with a dose dependent absorption ofPYY₃₋₃₆.

[0469] Additional Observations and Data:

[0470] Summary of findings:

[0471] At intranasal doses of 50 ug-200 ug, there is dose dependentplasma uptake of PYY.

[0472] The duration of elevated plasma concentrations is considerablylonger than would have been predicted, with an elimination half-lifecalculated at 55 minutes.

[0473] Cmax and AUC 0-t show good linearity with dose.

[0474] There is considerable inter-subject variability at a given dose.

[0475] Surprisingly, this study failed to detect postprandial elevationof PYY although the quantity of food actually eaten was not measured andif too little was eaten could explain the observations.

[0476] Visual-analog scale hunger questions suggest decreased hungerwith increasing doses of PYY.

[0477] Nausea and lightheadedness appear to be related to very highplasma concentration of PYY.

[0478] Notes:

[0479] In some cases pMol/L are used as the PYY measurement units; inother analyses, pg/mL are used. The conversion factor is pmol/L*4.05=pg/mL.

[0480] In some cases, the 150-minute time point is displayed in plots.Strictly speaking, this is a postprandial datapoint, and may thereforeconfound PK evaluation. However, an unexpected finding described in moredetail below is that the 30-minute postprandial timepoint is nodifferent from the baseline value.

[0481] PYY Plasma Concentrations:

[0482] The PYY assay described in this specification has been validatedfor its own PYY plasma concentration assay. Using this assay, samplesfrom each timepoint were assayed in triplicate. Note that out of the 180datapoints, 3 (1.6%) appear to be biologically implausible “outliers.”The data throughout this preliminary analysis use a dataset in whichthese three datapoints were removed.

Table 9

[0483] Descriptive PK Parameters: Calculated PK parameters include: AUC0-last AUC 0-inf Cmax (min * (min * Tmax (min) (pg/mL) pg/mL) pg/mL) T½(min) 20 60 47 3850 50 18 89 4960 12379 112 100 32 342 26535 41102 27150 23 530 31659 39476 39 200 23 683 34823 48618 42

[0484] Examination of the mean PK plots suggests a dose response from50-200 ug doses, but that the 20 μg dose is in the noise. Therefore,many of the subsequent analyses will be based on data only from the50-200 ug doses. We also propose that, because PYY is an endogenousmolecule, the AUC 0-t is more relevant than AUC 0-inf.

[0485] Tmax and T½ (Elimination Half Life): Tmax T_(1/2) (min) (min)Mean values for 50-200 ug dose groups: 24 55

[0486] The Tmax of 24 minutes is typical for a nasal product. Theelimination half life of 55 minutes is considerably longer than wouldhave been expected. Literature references indicated a t_(1/2) typicallyof 5-10 minutes. The elimination half-life may also be affected by somecontinued uptake from the nasal mucosa occurring after the Tmax and byformulation components that effect peptide metabolism. Alternatively,because the assay described in this specification employs an extractionprocedure, the assay will capture both free and protein-bound PYY,whereas an assay that does not use an extraction may assay primarily thefree fraction.

[0487] From this analysis of mean VAS change from baseline (mean of 10,30, and 60 minute values minus baseline) vs dose, one observes:

[0488] For VAS Q1 “How hungry do you feel?” subjects were less hungryafter receiving higher PYY doses. For VAS Q3, “How much do you think youcan eat?” subjects thought they could eat less after receiving higherdoses of PYY. However, for VAS Q2 “How full do you feel?” subjects feltless full after receiving higher doses of PYY. This suggests that thesensation follow PYY administration does not include fullness, bloating,or gastric hypercontractility.

EXAMPLE 8b PYY Human Administration and Weight Loss

[0489] The following PYY Nasal formulation was made. Reagent GradeVendor Cat # Lot # F.W. mg/ml % Cholorbutanol, anhydrous NF SpectrumCH123 RI1646 177.46 2.5 0.25 Methyl-β-Cyclodextrin Sigma C-4555 81K117945 4.5 L-α-Phospharidycholine Sigma P-7081 55H8377 565.7 1 0.1Didecanoyl Edetate Disodium USP Dow 1034N- 372.2 1 0.1 (EDTA) Chemicals00269-2 Sodium Citrate, Dihydrate USP Spectrum S0165 RH1056 294.1 1.60.16 Citric Acid, Anhydrous USP Sigma C-1857 062K003 192.13 0.9 0.09PYY(3-36), endotoxin-free Phoenix 059-02 420338 4049.71 2 0.2 PurifiedWater

[0490] Formulation pH 5+/−0.25

[0491] One or two sprays were administered daily to a human subject over10 day period and a weight loss of 2.5 pounds was recorded. Duringperiods ranging from 10 minutes to 12 hours after administration thesubject recorded reduced hunger.

EXAMPLE 9 Buccal Formulation of PYY3-36 (Prophetic)

[0492] Bilayer tablets are prepared in the following manner. An adhesivelayer is prepared by weighing 70 parts by weight polyethylene oxide(Polyox 30 IN; Union Carbide), 20 parts by weight polyacrylic acid(Carbopol 934P; B. F. Goodrich), and 10 parts by weight of acompressible xylitol/carboxymethyl cellulose filler (Xylitab 200;Xyrofin). These ingredients are mixed by rolling in ajar for 3 minutes.The mixture is then transferred to an evaporating dish and quickly wetgranulated with absolute ethanol to a semi-dough-like consistency. Thismass is immediately and rapidly forced through a 14 mesh (1.4 mmopening) stainless steel screen, to which the wet granules adhered. Thescreen is covered with perforated aluminum foil, and the wet granulesare dried overnight at 30° C. The dried granules are removed from thescreen and then passed through a 20 mesh (0.85 mm opening) screen tofurther reduce the size of the granules. Particles that do not passthrough the 20 mesh screen are ground briefly with a mortar and pestleto minimize the amount of fines and then passed through the 20 meshscreen. The resulting granules are then placed in a mixing jar, and 0.25parts by weight stearic acid and 0.06 parts by weight mint flavor(Universal Flavors) are added and blended to the granules. The finalpercentages by weight of the ingredients are thus 69.78% polyethyleneoxide, 9.97% compressible xylitol/carboxymethyl cellulose filler, 19.94%polyacrylic acid, 0.25% stearic acid, and 0.06% mint flavor. A 50 mgamount of this mixture is placed on a 0.375 inch diameter die andprecompressed on a Carver Press Model C with 0.25 metric ton pressurefor a 3 second dwell time to form the adhesive layer.

[0493] The active layer is prepared by weighing 49.39 parts by weight ofmannitol, 34.33 parts by weight of hydroxypropyl cellulose (Klucel L F;Aqualon, Wilmington, Del.) and 15.00 parts by weight of sodiumtaurocholate (Aldrich, Milwaukee, Wis.), and mixing by rolling in a jarfor 3 minutes. The mixture is then transferred to an evaporating dishand quickly wet granulated with absolute ethanol to a semi-dough-likeconsistency. This mass is immediately and rapidly forced through a 14mesh stainless steel screen, to which the wet granules adher. The screenis covered with perforated aluminum foil, and the granules dried at 30°C. The dried granulation is then passed sequentially through 20, 40(0.425 mm opening), and 60 (0.25 mm opening) mesh screens to reduceparticle size further. Particles that do not pass through a screen arebriefly ground with a mortar and pestle to minimize fines and thenpassed through the screen. The screened particles were weighed, and then0.91 parts by weight of PYY3-36 and 0.06 parts by weight of FD&C yellow#6HT aluminum lake dye are sequentially blended with the dry granulationby geometric dilution. The dyed granulation is then placed in a mixingjar and blended with 0.25 parts by weight magnesium stearate (lubricant)and 0.06 parts by weight mint flavor by rolling for 3 minutes. A 50 mgsample of this material is placed on top of the partially compressedadhesive layer and both layers are then compressed at 1.0 ton pressurefor a 3 second dwell time to yield a bilayer tablet suitable for buccaldelivery.

[0494] This procedure results in a gingival tablet wherein the activelayer contains 0.91% by weight of PYY3-36, 15% by weight of NaTC, and84.09% by weight of filler, lubricant, colorant, formulation aids, orflavoring agents.

EXAMPLE 10

[0495] A study was conducted comparing the ability of endotoxin-freePYY(3-36) (SEQ ID NO: 2) vs non-endotoxin-free PYY(3-36) to permeate thebronchial epithelium according of to the procedure of Example 1. It wasdetermined that about twice the amount of enodotoxin-free PYY(3-36)permeated the bronchial epithelium as compared to PYY(3-36) formulationthat contained endotoxin.

[0496] Both formulations contained Chlorobutanol 2.5 mg/ml, 2.mg/ml ofDDPC, 10 mg/ml of albumin, 1 mg/ml of EDTA (edetate disodium) and 45mg/ml of M-B-CD. One formulation contained endotoxin-free PYY(3-36) andthe other formulation contained 70 EUs or greater of endotoxin.

[0497] The average MTT of the PYY(3-36) formulation containing endotoxinwas 91.72% while the endotoxin-free PYY(3-36) formulation had an averageMTT of 100.16%.

[0498] The average permeation of the PYY(3-36) formulation containingendotoxin was 5.36%, while the average permeation of the endotoxin-freePYY(3-36) formulation was 10.75%.

[0499] A number of known mucosal delivery enhancing excipients can beeffectively combined with endotoxin-free Y2 receptor binding peptides,especially endotoxin-free PYY3-36, and can be used to improvenon-infusion formulations, especially oral delivery. Such excipients arecontained in the following patent applications that are incorporated byreference: U.S. patent applications 20030225300, 20030198658,20030133953, 20030078302, 20030045579, 20030012817, 20030012817,20030008900, 20020155993, 20020127202, 20020120009, 20020119910,20020065255, 20020052422, 20020040061, 20020028250, 20020013497,20020001591, 20010039258, 20010003001.

Oral Formulation of a Y2 Receptor-Binding Peptide

[0500] An oral formulation of a Y2 receptor-binding peptide can beprepared according to the following procedure. A preferred formulationfor oral delivery contains approximately 0.5 mg/kg endotoxin-free PYYand between 100 and about 200 mg/kg of one or more mucosal deliveryenhancing excipients.

(Prophetic) EXAMPLE 11

[0501] Preparation of N-cyclohexanoylphenylalanine Aldehyde:

[0502] Phenylalanine methyl ester (1 g., 0.0046 moles) is dissolved inpyridine 5 mL. Cyclohexanoyl chloride (0.62 mL) is added and the mixtureis stirred for 2 hours. The reaction mixture is poured onto hydrochloricacid (IN) and crushed ice. The aqueous mixture is extracted twice withtoluene. The combined toluene extracts are concentrated in vacuo to give1.1 g of crude N-cyclohexanoylphenylalanine methyl ester.

[0503] N-Cyclohexanoylphenylalanine methyl ester (0.5 g) is dissolved inethylene glycol dimethyl ether (20 mL). The solution is cooled to 70° C.and diisobutylaluminum hydride (2.04 mL of a 1.5M solution in toluene)is added. The resulting reaction mixture is stirred at −70° C. for 2hours. The reaction is quenched by dropwise addition of 2N hydrochloricacid. The mixture is extracted with cold ethyl acetate. The ethylacetate solution is washed with brine and dried over sodium sulfate.Concentration in vacuo furnishes a white solid, which is purified bysilica gel chromatography. ¹H NMR (300 MHz, DMSO-d₆): 9.5 (s, 1H), 8.2(d, 1H), 7.2 (m, 5H), 4.2 (m, 1H), 3.2 (d, 1H), 2.7 (d, 1H), 2.1 (m,1H), 1.6 (br.m, 4H), 1.2 (br.m, 6H). R (KBr): 3300, 3050, 2900, 2850,2800, 1700, 1600, 1500 cm-¹.

[0504] Mass Spec.: M+1 m/e 261.

EXAMPLE 12 Preparation of N-acetylphenylalanine Aldehyde:

[0505] N-Acetylphenylalanine methyl ester (4.2 g, 19 mmol) is dissolvedin ethylene glycol dimethyl ether. The solution is cooled to −70° C. anddiisobutylaluminum hydride (25.3 mL of a 1.5M solution in toluene, 39mmol) is added. The resulting reaction mixture is stirred at −70° C. for2 hours. The reaction is quenched by addition of 2N hydrochloric acid.The mixture is extracted 4 times with cold ethyl acetate and 4 timeswith toluene. The extracts are combined, washed with brine and driedover magnesium sulfate. Concentration in vacuo followed by silica gelchromatography furnishes 2.7 g of a white solid. The NMR is as reportedin the literature, Biochemistry, 18: 921-926 (1979).

EXAMPLE 13

[0506] Preparation of 3-acetamido-4-(p-hydroxy)phenyl-2-butanone(N-acetyltyrosinone):

[0507] A mixture of tyrosine (28.9 g, 16 mmol), acetic anhydride (97.9g, 96 mmol) and pyridine (35 g, 16 mmol) are heated to 100° C. for 1hour. The reaction mixture is concentrated in vacuo to furnish a yellowoil. The oil is distilled at reduced pressure to furnish 29.9 g or anoil.

[0508]¹H NMR (DMSO-d₆): NMR (d6-DMSO); 8.2 (d,1H), 7.3 (d, 2H), 7.0 (d,2H), 4.4 (m, 1H), 3.1 (dd, 1H), 2.7 (dd, 1H), 2.3 (s, 3H), 1.8 (s, 3H)

EXAMPLE 14

[0509] Preparation of 3-acetamido-7-amino-2-butanone (N-acetyllysinone):

[0510] Following the procedure of Example 3 lysine is converted toN-acetyllysinone.

[0511]¹H NMR (DMSO-d₆): 8.1 (d, 1H), 7.8 (br.m. 1H), 4.1 (m, 1H), 3.0(m, 2H), 2.0 (s, 3H), 1.9 (s, 3H) and 1.3 (br.m, 6H).

EXAMPLE 15

[0512] Preparation of 3-acetamido-5-methyl-2-butanone(N-acetylleucinone):

[0513] Following the procedure of Example 3 leucine is converted toN-acetylleucinone. ¹H NMR (DMSO-d6): 8.1 (d, 1H), 4.2 (m, 1H), 2.0 (s,3H), 1.8 (s, 3H), 0.8 (d, 6H).

EXAMPLE 16

[0514] Modification of 4-(4-aminophenyl)butyric Acid Using BenzeneSulfonyl Chloride

[0515] 4-(4-Aminophenyl)butyric acid, (20 g 0.11 moles) is dissolved in110 mL of aqueous 2N sodium hydroxide solution. After stirring for about5 minutes at room temperature, benzene sulfonyl chloride (14.2 mL, 0.11moles) is added dropwise into the amino acid solution over a 15 minuteperiod. After stirring for about 3 hours at room temperature the mixtureis acidified to pH 2 by addition of hydrochloric acid. This furnishes alight brown precipitate which is isolated by filtration. The precipitateis washed with warm water and dried. The melting point is 123-25° C.

[0516] If necessary, the modified amino acids can be purified byrecrystallization and/or chromatography.

EXAMPLE 17

[0517] Modification of 4-amindbenzoic Acid Using Benzene SulfonylChloride

[0518] Following the procedure of Example 64-aminobenzoic acid isconverted to 4-(phenylsulfonamido)benzoic acid.

EXAMPLE 18

[0519] Modification of 4-aminophenylacetic Acid, 4-aminohippuric Acid,and 4-aminomethylbenzoic Acid Using Benzene Sulfonyl Chloride Followingthe procedure of Example 6,4-aminophenylacetic acid, 4-aminohippuricacid, and 4-amino-methylbenzoic acid are converted to4-(phenylsulfonamido)phenylacetic acid, 4-(phenylsulfonamido)hippuricacid, and 4-(phenylsulfonamidomethyl)benzoic acid respectively.

EXAMPLE 19

[0520] Modification of Amino Acids with Benzene Sulfonyl Chloride

[0521] A mixture of sixteen amino acids are prepared prior to chemicalmodification. The constituents of the mixture are summarized in theTable below. 65 grams of the amino acid mixture (total concentration of[—NH₂] groups=0.61 moles) is dissolved in 760 mL of 1N sodium hydroxidesolution (0.7625 equivalents) at room temperature. After stirring for 20minutes, benzene sulfonyl chloride (78 ml, 1 eq.) is added over a 20minute period. The reaction mixture is then stirred for 2.5 hours,without heating. As some precipitation may occur, additional NaOHsolution (2N) may be added to the solution until it reaches pH 9.3. Thereaction mixture is stirred overnight at room temperature. Thereafter,the mixture is acidified using dilute hydrochloric acid (38%, 1:4) and acream colored material precipitates out. The resulting precipitate isisolated by decantation and dissolved in sodium hydroxide (2N). Thissolution is then reduced in vacuo to give a yellow solid, which is driedon the lyophilizer. TABLE 10 Amino Acid Composition No. of moles % ofTotal of each Amino No. of Moles Amino Acid Weight (g) Weight Acid(×10⁻²) of - [—NH₂] Thr 2.47 3.8 2.07 2.07 Ser 2.25 3.46 2.1 2.1 Ala4.61 7.1 5.17 5.17 Val 4.39 6.76 3.75 3.75 Met 0.53 0.82 0.35 0.35 Ile2.47 3.8 0.36 0.36 Leu 3.86 5.94 2.95 2.95 Tyr 1.03 1.58 0.56 0.56 Phe4.39 6.76 0.27 0.27 His 2.47 3.8 1.6 3.2 Lys 4.94 7.6 3.4 6.8 Arg 5.137.9 2.95 5.90 Glutamine 9.87 15.18 6.76 13.42 Glutamic 9.87 15.18 6.706.70 Acid Asparagine 3.32 5.11 2.51 5.02 Aspartic 3.32 5.11 2.50 2.50Acid

EXAMPLE 20

[0522] Modification of a Mixture of Five Amino Acids Using BenzeneSulfoniyl Chloride

[0523] An 86.1 g (0.85 moles of NH2) mixture of amino acids (see Tablebelow) is dissolved in 643 mL (1.5 eq.) of aqueous 2N sodium hydroxidesolution. After stirring for 30 minutes at room temperature, benzenesulfonyl chloride (108 mL, 0.86 moles) is added portionwise into theamino acid solution over a 15 minute period. After stirring for 2.5hours at room temperature, the pH of the reaction mixture (pH 5) isadjusted to pH 9 with additional 2N sodium hydroxide solution. Thereaction mixture is stirred overnight at room temperature. Thereafter,the pH of the reaction mixture is adjusted to pH 2.5 by addition ofdilute aqueous hydrochloric acid solution (4: 1, H₂O: HCl) and aprecipitate of modified amino acids is formed. The upper layer isdiscarded and the resulting yellow precipitate is isolated bydecantation, washed with water and dissolved in 2N sodium hydroxide(2N). The solution is reduced in vacuo to give a yellow solid, which islyophilized overnight. TABLE 11 Moles of Amino Acid Moles of Amino Acid(×10⁻²) [—NH₂] × 10⁻² Valine 7.5 7.5 Leucine 10.7 10.5 Phenylalanine13.4 13.4 Lysine 21.0 42.0 Arginine 6.0 12.0

EXAMPLE 21

[0524] Modification of a Mixture of Five Amino Acids Using BenzoylChloride

[0525] An 86 g (0.85 moles of NH₂) mixture of amino acids (see Table inExample 20) is dissolved in 637 mL (1.5 eq.) of aqueous 2N sodiumhydroxide solution. After stirring for 10 minutes at room temperature,benzoyl chloride (99 mL, 0.85 moles) is added portionwise into the aminoacid solution over a 10 minute period. After stirring for 2.5 hours atroom temperature, the pH of the reaction mixture (pH 12) is adjusted topH 2.5 using dilute hydrochloric acid (4: 1, H₂O: HCl) and a precipitateof modified amino acids is formed. After settling for 1 hour, theresulting precipitate is isolated by decantation, washed with water anddissolved in sodium hydroxide (2N). This solution is then reduced invacuo to give crude modified amino acids as a white solid (expectedyield 220.5 g).

EXAMPLE 22 Modification of L-valine Using Benzene Sulfonyl Chloride

[0526] L-Valine (50 g, 0.43 mol) is dissolved in 376 mL (0.75 eq.) ofaqueous 2N sodium hydroxide by stirring at room temperature for 10minutes. Benzene sulfonyl chloride (68.7 mL, 0.38 mol, 1.25 eq.) is thenadded to the amino acid solution over a 20 minute period at roomtemperature. After stirring for 2 hours at room temperature, aprecipitate appears. The precipitate is dissolved by adding 200 mL ofadditional 2N sodium hydroxide solution. After stirring for anadditional 30 minutes, dilute aqueous hydrochloric acid solution (4: 1,H₂O: HCl) is added until the pH of the reaction mixture reaches 2.6. Aprecipitate of modified amino acids formed and is recovered bydecantation. This material is dissolved in 2N sodium hydroxide and driedin vacuo co to give a white solid. Expected yield of crude modifiedamino acids is 84.6 g, 77%).

EXAMPLE 23 Modification of Phenylalanine Methyl Ester Using HippurylChloride

[0527] L-Phenylalanine Methyl Ester Hydrochloride (15 g, 0.084 mole) isdissolved in dimethylformamide (DMF) (100 mL) and to this is addedpyridine (30 mL). A solution of hippuryl chloride (16.6 g, 0084 moles in100 mL DMF) is immediately added to the amino acid ester solution in twoportions. The reaction mixture is stirred at room temperature overnight.The reaction mixture is then reduced in vacuo and dissolved in 1Naqueous sodium hydroxide. The solution is heated at 70° C. for 3 hoursin order to hydrolyze the methyl ester to a free carboxyl group.Thereafter, the solution is acidified to pH 2.25 using dilute aqueoushydrochloric acid solution (1:3 HCl/H₂O). A gum-like precipitate isformed and this is recovered and dissolved in 1N sodium hydroxide. Thesolution is reduced in vacuo to afford an expected 18.6 g of crudemodified amino acid product. After recrystallization from acetonitrile,pure modified phenylalanine (expected yield 12 g) is recovered as awhite powder. m.p. 223-225° C.

EXAMPLE 24 Preparation of Dosing Solutions of PYY(3-36)

[0528] In a test tube 568 mg of acetyl phenylalanine aldehyde, 132 mg ofcarbomethoxy phenylalanylleucine and 100 mg acetyl-Phe-Leu-Leu-Argaldehyde are added to 2.9 ml of 15% ethanol. The solution is stirred andNaOH (1.0 N) is added to raise the pH to 7.2. Water is added to bringthe total volume to 4.0 mL. The sample had a carrier concentration of200 mg/mL. PYY(3-36) (800 μg) is added to the solution. The totalPYY3-36 concentration is 200 μg/mL.

[0529] Following a similar procedure a second solution having 668 mg ofacetyl phenylalanine aldehyde and 132 mg of carbomethoxyphenylalanylleucine as the carrier composition and a third solutionhaving as the carrier acetyl phenylalanine aldehyde are prepared. Eachsolution had an endotoxin-free PYY(3-36) concentration of 200 μg/mL.

EXAMPLE 25 Preparation of Modified Amino Acid/PYY(3-36) Compositions

[0530] Preparation of Modified Amino Acid Microspheres ContainingEncapsulatedendotoxin-free PYY3-36

[0531] The modified amino acid mixture, prepared in accordance withExample 9, is dissolved at 40° C. in distilled water (pH 7.2) at aconcentration of 100 mg/ml. The solution is then filtered with a 0.2micron filter and the temperature is maintained at 40° C. PYY3-36(Bachem) is dissolved in an aqueous solution of citric acid (1.7N) andgelatin (5%) at a concentration of 150 mg/ml. This solution is thenheated to 40 C. The two heated solutions are then mixed 1:1 (v/v). Theresulting microsphere suspension is then filtered with glass wool andcentrifuged for 50 minutes at 1000 g. The pellet is resuspended with0.85N citric acid to a volume 5 to 7 fold less than the original volume.PYY3-36 concentration of the resuspended pellet is determined by HPLC.Additional microspheres are made according to the above procedurewithout PYY3-36. These “empty microspheres” are used to dilute theencapsulated salmon PYY3-36 microsphere preparation to a final dosingsuspension, if needed.

[0532] (b) Preparation of a Soluble Modified Amino Acid Carrier/PYY3-36System

[0533] A soluble amino acid dosing preparation containing PYY3-36 isprepared by dissolving the modified amino acid material in distilledwater (pH 8) to an appropriate concentration. The solution is heated to40° C. and then filtered with a 0.2 micron filter. PYY3-36, alsodissolved in distilled water, is then added to the modified amino acidsolution prior to oral administration.

Pulmonary Delivery of PYY3-36 (Prophetic)

[0534] The carrier compounds, prepared as described below may be useddirectly as a delivery carrier by simply mixing one or more compound orsalt, poly amino acid or peptide with an endotoxin-free Y2receptor-binding peptide for pulmonary delivery.

[0535] The administration mixtures are prepared by mixing an aqueoussolution of the carrier with an aqueous solution of the activeingredient, just prior to administration. Alternatively, the carrier andthe biologically or chemically active ingredient can be admixed duringthe manufacturing process. The solutions may optionally containadditives such as phosphate buffer salts, citric acid, acetic acid,gelatin, and gum acacia.

[0536] A number of known pulmonary delivery methods can useendotoxin-free Y2 receptor-binding peptides, especially PYY3-36, toimprove the delivery of PYY to the lungs. The following non-limitingpatent applications are incorporated herein by reference for pulmonarydelivery: U.S. patent application 20030223939, 20030215514, 20030215512,20030209243, 20030203036, 20030198601, 20030183228, 200301885765,20030150454, 20030124193, 20030094173.

EXAMPLE 26 Preparation of Carriers

[0537] Preparation of 2-(4-(N-salicyloyl)aminophenyl) propionic acid(Carrier B)

[0538] A slurry of 58.6 g (0.355 mol) of 2-(4-aminophenyl)propionic acidand 500 ml of methylene chloride is treated with 90.11 ml (77.13 g.0-710 mol) of trimethylsilyl chloride and is heated to reflux for 120min. The reaction mixture is cooled to 0° C. and treated with 184.44 ml(107.77 g, 1.065 mol) of triethylamine. After stirring for 5 minutes,this mixture is treated with a solution of 70.45 g (0.355 mol) ofO-acetylsalicyloyl chloride and 150 ml of methylene chloride. Thereaction mixture is warmed to 25° C. and stirred for 64 hr. Thevolatiles are removed in vacuo. The residue is stirred in 2N aqueoussodium hydroxide for one hour and acidified with 2 M aqueous sulfuricacid. The solid is recrystallized twice from ethanol/water to give a tansolid. Isolation by filtration results in an expected yield of 53.05 g(52% yield) of 2-(4-(N-salicyloyl)aminophenyl)propionic acid.Properties. Solubility: 200 mg/m: 200 mg+350.μL 2N NaOH+650.μLH₂O-pH-7.67. Analysis: C, 67.36; H, 5.3; N, 4.91 . . . .

[0539] Preparation of Sodium 2-(4-(N-salicyloyl)aminophenyl)propionate(Sodium Salt of Carrier B)

[0540] A solution of 53.05 g (0.186 mol) of2-(4-(N-salicyloyl)aminophenyl-)propionic acid and 300 ml of ethanol istreated with 7.59 g (0.190 mol) of NaOH dissolved in 22 ml of water. Thereaction mixture is stirred for 30 min at 25° C. and for 30 min at 0° C.The resulting pale yellow solid is isolated by filtration to give 52.61g of sodium 2-(4-(N-salicyloyl)aminophenyl)propionate. Properties.Solubility: 200 mg/ml clear solution, pH=6.85. Analysis C, 60.45; H,5.45; N, 3.92; Na, 6.43. Melting point 236-238° C.

[0541] Preparation of the Sodium Salt of Carrier C

[0542] A 2L round bottom flask equipped with a magnetic stirrer and areflux condenser is charged with a suspension of3-(4-aminophenyl)propio-nic acid (15.0 g. 0.084 moles. 1.0 equiv.) indichloromethane (250 ml). Chlorotrimethylsilane (18.19 g, 0.856 moles,2.0 equiv.) is added in one portion, and the mixture is heated to refluxfor 1.5 h under argon. The reaction is allowed to cool to roomtemperature and is placed in an ice bath (internal temperature <10° C.).The reflux condenser is replaced with an addition funnel containingtriethylamine (25.41 g, 0.251 moles, 3.0 equiv.). The triethylamine isadded dropwise over 15 min, and a yellow solid forms during theaddition. The funnel is replaced by another addition funnel containing asolution of 2,3-dimethoxybenzoylchlo-ride (I 8.31 g, 0.091 moles, 1.09equiv.) in dichloromethane (100 mL). The solution is added dropwise over30 nm. The reaction is stirred in the ice bath for another 30 min and atambient temperature for 3 h. The dicholoromethane is evaporated in vacuoto give a brown oil. The brown oil is cooled in an ice bath, and anice-cold solution of saturated sodium bicarbonate (250 ml) is added. Theice bath is removed, and the reaction is stirred 1 h to afford a clearbrown solution. The solution is acidified with concentrated HCl andstored at ca SC for 1 hour. The mixture is extracted withdichloromethane (3.times. 100 mL), dried over sodium sulfate, the saltsfiltered off and the dichloromethane removed in vacuo. The resultingsolid is recrystallized from 50% ethyl acetate/water (v/v) to affordCarrier C acid as off white needles (25.92 g. 90%). Analysis forC₁₉H₂₁NO₅: C, 66.46; H, 6.16; N, 4.08. mp=99-102° C.

[0543] 12 grams of the Carrier C acid is dissolved in ethanol, 75 mL,with warming. To this solution a 8.5 M Sodium hydroxide (1.02 molarequivalents, 1.426 grams in 4.5 mL water) solution is added. The mixtureis stirred for 15 minutes. Approximately three quarters of the ethanolis remove in vacuo and n-heptane, 100 mL, is added to the resulting oilcausing a precipitate to form. The solids are dried in vacuo at 50° C.Analysis: C₁₉H₂₀NO₅Na0.067H₂O: C, 62.25; H, 5.54; N, 3.82; Na, 6.27.

[0544] Preparation of N-(4-methylsalicyloyl)-8-aminocaprylic acid(Carrier D)

[0545] (a) Preparation of Oligo(4-methylsalicylate)

[0546] Acetic anhydride (32 mL, 34.5 g, 0.338 mol, 1.03 eq),4-methylsalicylic acid (50 g, 0.329 mmol, 1.00 eq), and xylenes (100 mL)are added to a 1 L, four-neck flask fitted with a magnetic stir bar, athermometer, and a condenser. The flask is placed in a sand bath andheating of the cloudy white mixture begun. The reaction mixture clearsto a yellow solution around 90° C. Most of the volatile organics(xylenes and acetic acid) are distilled into the Dean-Stark trap overthree hours (135-146° C.). Distillation is continued for another hour (atotal of 110 mL distilled), during which the pot temperature slowlyrises to 204° C. and the distillate slows to a trickle. The residue ispoured off while still hot into an aluminum tray. Upon cooling a brittleyellow glass forms. The solid is ground to a fine powder. Theoligo(4-methylsalicylate) received is used without further purification.

[0547] (b) Preparation of N-(4-methylsalicyloyl)-8-aminocaprylic Acid

[0548] A 7M solution of potassium carbonate (45 mL, 43.2 g, 0.313 mol,0.95 eq), 8-aminocaprylic acid (41.8 g, 262 mol, 798 eq), and water (20mL) are added to a 1 L round bottom flask equipped with a magnetic stirbar, condenser, and an addition fuel. The white cloudy mixture istreated with a solution of oligo(4-methylsalicylate) (44.7 g, 0.329 mmol1.0 eq) and dioxane (250 mL), added over thirty minutes. The reactionmixture is heated to 90° C. for 3 hours (at which time the reaction isdetermined to have finished, by HPLC). The clear orange reaction mixtureis cooled to 30° C. and acidified to pH=2 with 50% aqueous sulfuric acid(64 g). The resulting solid is isolated by filtration. The white solidis recrystallized from 1170 mL of 50% ethanol-water. The solid isrecovered by filtration and is dried over 18 hours in a 50° C. vacuumoven. The N-(4-methylsalicyloyl)-8-ami-nocaprylic acid is isolated as awhite solid (30.88 g, 52%); mp=113-114°. Analysis: C₆H₂₃NO₄: C, 65.51;H, 7.90; N, 4.77.

[0549] An aqueous solution of PYY(3-36) is then prepared and mixed withone or more of the carrier to produce a PYY(3-36) composition, whichthen can be sprayed into the lungs. A suitable concentration of PYY3-36for the resultant composition should be about 400 μg/mL. See U.S. patentapplication No. 20030072740.

EXAMPLE 27 Total Extraction Radioimmunoassay for the Determination ofthe concentration of PYY in Plasma

[0550] 1.0 Introduction:

[0551] A radioimmunoassay was developed to measure the concentration ofHuman Peptide YY(3-36) (hPYY) in human plasma. Samples are collectedwith anticoagulant (EDTA) and protease inhibitor (aprotinin) and frozen.The assay is a four day process. Samples, controls, and standards areextracted in alcohol and dried on Day 1. All samples are reconstitutedand mixed with a polyclonal rabbit antiserum directed against hPYY onDay 2. Iodinated hPYY is added on Day 3. Specific precipitating agents(Goat anti-Rabbit IgG and Normal Rabbit Serum) are added on Day 4. Boundtracer is separated from free tracer by centrifugation, and the boundtracer is counted in the gamma counter. Concentration is calculated byinterpolation of a standard curve and assay performance is controlledwith Quality Control samples.

[0552] 2.0 Materials:

[0553] 2.1 Peninsula PYY kit (Peninsula Laboratories, Cat. No.S-2043-0001)

[0554] 2.2 Reagent Alcohol (Fisher Inc., Cat. No. A995-4) (orequivalent)

[0555] 2.3 Stripped human plasma (with Lithium Heparin, fasted, pooled)Golden West Biologics Inc. (Cat. No., SD1020-H) (Analytical SOP # A-003)

[0556] 2.4 Ice Baths (Fisher, Cat No. 11-676-36) (or equivalent)

[0557] 2.5 Disposable 10 mL pipets (Fisher Cat. No. 13-678-11E) (orequivalent)

[0558] 2.6 Standard Synthetic Human PYY from Nastech QC (3-36) (BachemCat. No. H8585)

[0559] 2.7 Distilled Water (Milli-Q Millipore, Cat. No. ZMQ56VFTI) (orequivalent)

[0560] 2.8 Triton X-100 (Sigma, Cat. No. T-9284) (or equivalent)

[0561] 2.9 Aluminum Foil (Fisher, Cat. No. 01-213-3) (or equivalent)

[0562] 2.10 Aprotinin (ICN Biomedicals Inc. Cat. No. 190779) (orequivalent)

[0563] 2.11 12×75 mm tubes (Evergreen Scientific, Cat. No. 214-2023-010)(or equivalent)

[0564] 2.12 12×75 mm tube caps (Evergreen Scientific, Cat. No.300-2912-G20) (or equivalent)

[0565] 2.13 1.5 mL microfuge tubes (Fisher, Cat. No. 05402-25) (orequivalent)

[0566] 2.14

[0567] 3.0 Instruments:

[0568] 3.1 Wallac WIZARD 1470 Automatic Gamma Counter (Perkin Elmer,Model No. 1470-002) (or equivalent)

[0569] 3.2 Isotemp Basic Freezer, −70° C. (Kendro Laboratory Products,Model No.C90-3A31) (or equivalent)

[0570] 3.3 CentriVap Concentrator (Labconco, Cat. No. 7810000) (orequivalent)

[0571] 3.4 VX-2500 Multi-tube Vortexer (VWR, Cat. No. 58816-115) (orequivalent)

[0572] 3.5 Marathon 21000R Centrifuge (Fisher, Cat. No. 04-977-21000R)(or equivalent)

[0573] 3.6 Swinging bucket rotor (Fisher, Cat. No. 04-976-006) (orequivalent)

[0574] 3.7 Motorized pipet-aid (Fisher, Cat. No. 13-681-15E) (orequivalent)

[0575] 3.8 Eppendorf Micropipette

[0576] 3.8.1 2 μL-20 μL (Fisher, Cat. No. 21-371-6) (or equivalent)

[0577] 3.8.2 20 μL-200 μL (Fisher, Cat. No.21-371-10) (or equivalent)

[0578] 3.8.3 100 μL-1000 μL (Fisher, Cat. No.21-371-13) (or equivalent)

[0579] 3.9 Eppendorf Repeating Pipettor (Fisher, Cat. No. 21-380-9) (orequivalent)

[0580] 3.10 Eppendorf Repeating Pipettor Combi-tips

[0581] 3.10.1 2.5 mL (Fisher, Cat. No.21-381-331) (or equivalent)

[0582] 3.10.2 25 mL (Fisher, Cat. No.21-381-115) (or equivalent)

[0583] 3.11 Positive displacement pipet (Fisher, Cat. No. 21-169-10A)(or equivalent)

[0584] 4.0 Procedure

[0585] Day 1

[0586] 4.1 Thaw necessary reagents and samples for the assay. PrepareRIA buffer to 1× concentration (RIAB) if sufficient amount is notavailable.

[0587] 4.2 Prepare standard curve samples in pooled stripped humanplasma. Prepare as follows if using a starting concentration of 12.8μg/mL.

[0588] 4.2.1 Add 990 μL RIAB to tube 0.

[0589] 4.2.2 Add 990 μL pooled plasma to tube A.

[0590] 4.2.3 Add 500 μL pooled plasma to tubes B-H.

[0591] 4.2.4 Add 10 μL 12.8 μg/mL Standard to tube O. Vortex.

[0592] 4.2.5 Add 10 μL solution from tube 0 to tube A. Vortex.

[0593] 4.2.6 Add 500 pL solution from tube A to tube B. Vortex.

[0594] 4.2.7 Add 500 μL solution from tube B to tube C. Vortex.

[0595] 4.2.8 Repeat dilutions as in 4.2.7 through tube H. (See Diagram#1)

[0596] 4.3 Dilute unknown human plasma samples to be tested ifnecessary. Samples should be diluted in pooled stripped human plasma.

[0597] 4.4 Add 1.2 mL of cold alcohol to empty tubes for NSB, TB, allStandards, QC samples, and human plasma samples to be tested.

[0598] 4.5 Add 400 μL of pooled stripped human plasma to NSB and TBtubes. Cap, Vortex.

[0599] 4.6 Add 400 μL of each prepared Standard sample from 4.2.5 to4.2.8 to respective standard curve tubes H-A (See Diagram #1). Cap,Vortex.

[0600] 4.7 Add 400 μL of QC samples to respective tubes. Cap, Vortex.

[0601] 4.8 Add 400 μL of each sample to be tested its respective tube.Cap, Vortex.

[0602] 4.9 Incubate all samples on ice for 30-60 minutes.

[0603] 4.10 Turn on the cold-trap switch on the Concentrator.

[0604] 4.11 Centrifuge all tubes at 3000 rpm, 4° C. for 15 minutes.

[0605] 4.12 Transfer 1.3 mL of supernatant from each sample to a new setof empty tubes. Store in an ice bath or at 2-8° C. if not spunimmediately.

[0606] 4.13 Place samples in the Concentrator.

[0607] 4.14 Samples should spin for two hours at 40° C., then at ambienttemperature for a total of 5 hours or until dry.

[0608] 4.15 Remove dried samples, cover and store overnight at 2-8° C.

[0609] Day 2

[0610] 4.16 Remove the dried tubes from the 2-8° C. cooler.

[0611] 4.17 Add 100 μL of 4× RIA buffer concentrate to each tube.

[0612] 4.18 Add 100 μL of 0.6% TX100 to each tube. (Attachment #1)Vortex for a minimum of 30 seconds to ensure all extracts are fullyreconstituted.

[0613] 4.19 Incubate all samples on ice for 30-60 minutes.

[0614] 4.20 Add 200 μL of distilled water to each tube. Vortex.

[0615] 4.21 Transfer 100 μL of each sample extract to respective tube.

[0616] Note: NSB, TB, TC, Standard Curve samples, and QCs are typicallyrun in triplicate, requiring three tubes per sample. Human plasmasamples many be tested in any variation (up to three replicates)depending on sample availability.

[0617] 4.22 Prepare Rabbit anti-PYY as described in the PeninsulaLaboratories kit insert.

[0618] 4.23 Add 100 μL RIAB to each NSB tube.

[0619] 4.24 Add 200 μL RIAB to each TC tube.

[0620] 4.25 Add 100 μL Rabbit anti-PYY to all remaining tubes. Vortex.

[0621] 4.26 Cover with foil and store overnight at 2-8° C.

[0622] Day 3

[0623] 4.27 Remove the tubes from the 2-8° C. cooler.

[0624] 4.28 Prepare ¹²⁵I-Peptide YY tracer (Attachment #2).

[0625] 4.29 Add 100 μL of prepared tracer to all tubes. Cap and vortex.

[0626] 4.30 Store overnight at 2-8° C.

[0627] Day 4

[0628] 4.31 Remove the tubes from the 2-8° C. cooler.

[0629] 4.32 Prepare Goat anti-Rabbit IgG serum (GARGG) and Normal RabbitSerum (NRS) as described in the Peninsula Laboratories kit insert.

[0630] 4.33 Add 100 μL GARGG to each tube (except TC tubes).

[0631] 4.34 Add 100 μL NRS to each tube (except TC tubes). Vortex.

[0632] 4.35 Incubate 90-120 minutes at room temperature.

[0633] 4.36 Add 500 μL RIAB to tubes to be centrifuged immediately(except TC tubes). Vortex.

[0634] Note: 500 μL RIAB should be added to tubes just prior tocentrifugation. Only add RIAB to the number of tubes that are ready tobe centrifuged. 500 μL RIAB should be added to additional tubes whenthey are ready to be centrifuged.

[0635] 4.37 Centrifuge tubes (containing 500 μL RIAB) at 3000 rpm at 4°C., for 15 minutes. Do not centrifuge TC tubes.

[0636] 4.38 Aspirate supernatant from centrifuged tubes.

[0637] 4.39 Place tubes in designated black racks for counting on theGamma counter. The first rack should have the appropriate Program numberattached. All racks that follow should contain no program number.Samples should be added in the following order:

[0638]4.39.1 NSB tubes

[0639] 4.39.2 TB tubes

[0640] 4.39.3 TC tubes

[0641] 4.39.4 Standard tubes (increasing concentration)

[0642] 4.39.5 QC samples (3 concentrations)

[0643] 4.39.6 Unknown human samples

[0644] 4.39.7 QC samples (3 concentrations)

[0645] 4.40 Place an empty black rack with the Stop label attached afterall samples to be counted.

[0646] 4.41 Press ‘Start’ on the Gamma Counter keypad to start counting.

[0647] 4.42 Press ‘E’ for enter on the Gamma Counter keypad to displayCPM results.

[0648] 5.0 Evaluation of Results

[0649] 5.1 The following guidelines are applied to the identificationand rejection of outliers in the assay. In order for a result to qualifyas an outlier and not be included in the final calculation of results,all of the following conditions must be met.

[0650] 5.1.1 QCs and unknown samples:

[0651] 5.1.1.1% CV of all replicates must be great than 20%.

[0652] 5.1.1.2 There must be at least three results to evaluate.

[0653] 5.1.1.3 The difference between the suspected outlier and theresult next closest in value must be greater than 20%.

[0654] 5.1.1.4 The difference between the high and low remaining resultsmust be less than 20%.

[0655] 5.1.2 Standard Curve samples:

[0656] 5.1.2.1% CV of all replicates much be greater than 15%.

[0657] 5.1.2.2 There must be at least three results to evaluate.

[0658] 5.1.2.3 The difference between the suspected outlier and theresult next closest in value must be greater than 15%.

[0659] 5.1.2.4 The difference between the high and low remaining resultsmust be less than 15%.

[0660] 6.0 Assay Specifications

[0661] 6.1 QC samples are prepared at the following concentrations. TwoQC samples at each concentration are tested in an assay. Four of the sixQC samples tested must be within the following ranges (±30% of nominalconcentration). At least one of the two QCs tested at any concentrationmust be within range of the assay for data to be acceptable.

[0662] 6.1.1 QC1 (100 pg/mL) 70-130 pg/mL

[0663] 6.1.2 QC2 (200 pg/mL) 140-260 pg/mL

[0664] 6.1.3 QC3 (500 pg/mL) 350-650 pg/mL

[0665] 6.2 Standard curve parameter requirements TBD.

[0666] PYY RIA Standard: Tube designation Concentration of Standard A1280 pg/mL B  640 pg/mL C  320 pg/mL D  160 pg/mL E  80 pg/mL F  40pg/mL G  20 pg/mL H  10 pg/mL

[0667] Attachment #1

0.6% TX-100

[0668] Reagent: 0.6% TX-100

[0669] Materials: Milli-Q Distilled Water

[0670] TX-100

[0671] Preparation: 1) Measure 50 mL of Milli-Q Distilled Water

[0672] 2) Add 300 μL of TX-100 using positive displacement pipet

[0673] 3) Mix well.

[0674] Attachment #2

¹²⁵-Peptide PYY Tracer

[0675] Reagent: ¹²⁵I-Peptide PYY Tracer

[0676] Materials: 1× RIA Buffer

[0677]¹²⁵I-Peptide PYY

[0678] Preparation: 1) Reconstitute tracer with 1 mL of 1× RIA Buffer.

[0679] 2) Measure the quantity of the tracer on the Gamma Counter.Transfer 10 μL of reconstituted tracer to a tube. Place it in a blackrack for the Gamma Counter with Program #30 attached.

[0680] 3) Place rack on the Gamma Counter with the Stop rack behind it.

[0681] 4) Press ‘Start” to begin counting, then ‘E’ to view CPM results.

[0682] 5) Determine amount of tracer (X μL) to prepare and RIAB (Y mL)needed as follows:${X\quad {µL}} = \frac{( {5\quad {µL}} )\quad ( {{cpm}\quad {value}} )( {{\# \quad {tubes}} + 10} )}{( {{cpm}\quad {from}\quad {stock}\quad {solution}} )}$

 Y mL=(0.1) (# tubes+10)

[0683] 6) Combine X μL of ¹²⁵I-Peptide YY with Y mL of RIAB. Mix well.

EXAMPLE 28 Preparation of an NPY Formulation Free of a Stabilizer thatis a Protein

[0684] A PYY formulation suitable for intranasal administration of NPY,which is substantially free of a stabilizer that is a protein isprepared having the formulation listed below.

[0685] 1. About ¾ of the water is added to a beaker and stirred with astir bar on a stir plate and the sodium citrate is added until it iscompletely dissolved.

[0686] 2. The EDTA is then added and stirred until it is completelydissolved.

[0687] 3. The citric acid is then added and stirred until it iscompletely dissolved.

[0688] 4. The methyl-β-cyclodextrin is added and stirred until it iscompletely dissolved.

[0689] 5. The DDPC is then added and stirred until it is completelydissolved.

[0690] 6. The lactose is then added and stirred until it is completelydissolved.

[0691] 7. The sorbitol is then added and stirred until it is completelydissolved.

[0692] 8. The chlorobutanol is then added and stirred until it iscompletely dissolved.

[0693] 9. The NPY(3-36) is added and stirred gently until it dissolved.

[0694] 10. Check the pH to make sure it is 5.0±0.25. Add dilute HCl ordilute NaOH to adjust the pH.

[0695] 11. Add water to final volume. TABLE 12 Reagent Grade Vendormg/mL % Cholorbutanol, anhydrous NF Spectrum 5.0 0.50Methyl-β-Cyclodextrin Sigma 45 4.5 L-α-Phospharidycholine Sigma 1 0.1Didecanoyl Edetate Disodium (EDTA) USP Dow Chemicals 1 0.1 SodiumCitrate, Dihydrate USP Spectrum 1.62 0.162 Citric Acid, Anhydrous USPSigma 0.86 0.086 α-Lactose monohydrate Sigma 9 0.9 Sorbitol Sigma 18.21.82 NPY(3-36) GMP Bachem 1 0.1 Purified Water

EXAMPLE 29

[0696] A second formulation is prepared as above, except theconcentration of NPY(3-36) is 15 mg/mL as shown below in Table 13. TABLE13 Reagent Grade Vendor mg/ml % Cholorbutanol, anhydrous NF Spectrum 5.00.50 Methyl-β-Cyclodextrin Sigma 45 4.5 L-α-Phospharidycholine Sigma 10.1 Didecanoyl Edetate Disodium USP Dow Chemicals 1 0.1 Sodium Citrate,Dihydrate USP Spectrum 1.62 0.162 Citric Acid, Anhydrous USP Sigma 0.860.086 α-Lactose monohydrate Sigma 9 0.9 Sorbitol Sigma 18.2 1.82NPY(3-36) GMP Bachem 15 0.1 Purified Water

EXAMPLE 30 Preparation of Pancreatic Pepetide (PP) Formulation Free of aStabilizer that is a Protein

[0697] A PYY formulation suitable for intranasal administration of PP,which is substantially free of a stabilizer that is a protein isprepared having the formulation listed below.

[0698] 1. About ¾ of the water is added to a beaker and stirred with astir bar on a stir plate and the sodium citrate is added until it iscompletely dissolved.

[0699] 2. The EDTA is then added and stirred until it is completelydissolved.

[0700] 3. The citric acid is then added and stirred until it iscompletely dissolved.

[0701] 4. The methyl-β-cyclodextrin is added and stirred until it iscompletely dissolved.

[0702] 5. The DDPC is then added and stirred until it is completelydissolved.

[0703] 6. The lactose is then added and stirred until it is completelydissolved.

[0704] 7. The sorbitol is then added and stirred until it is completelydissolved.

[0705] 8. The chlorobutanol is then added and stirred until it iscompletely dissolved.

[0706] 9. The PP(3-36) is added and stirred gently until it dissolved.10. 11 Check the pH to make sure it is 5.0±0.25. Add dilute HCl ordilute NaOH to adjust the pH.

[0707] 11. Add water to final volume. TABLE 14 Reagent Grade Vendormg/mL % Cholorbutanol, anhydrous NF Spectrum 5.0 0.50Methyl-β-Cyclodextrin Sigma 45 4.5 L-α-Phospharidycholine Sigma 1 0.1Didecanoyl Edetate Disodium USP Dow Chemicals 1 0.1 Sodium Citrate,Dihydrate USP Spectrum 1.62 0.162 Citric Acid, Anhydrous USP Sigma 0.860.086 α-Lactose monohydrate Sigma 9 0.9 Sorbitol Sigma 18.2 1.82PP(3-36) GMP Bachem 1 0.1 Purified Water

EXAMPLE 31

[0708] A second formulation is prepared as above, except theconcentration of PP(3-36) is 15 mg/mL as shown below in Table 15. TABLE15 Reagent Grade Vendor mg/ml % Cholorbutanol, anhydrous NF Spectrum 5.00.50 Methyl-β-Cyclodextrin Sigma 45 4.5 L-α-Phospharidycholine Sigma 10.1 Didecanoyl Edetate Disodium USP Dow Chemicals 1 0.1 Sodium Citrate,Dihydrate USP Spectrum 1.62 0.162 Citric Acid, Anhydrous USP Sigma 0.860.086 α-Lactose monohydrate Sigma 9 0.9 Sorbitol Sigma 18.2 1.82PP(3-36) GMP Bachem 15 0.1 Purified Water

[0709] Although the foregoing invention has been described in detail byway of example for purposes of clarity of understanding, it will beapparent to the artisan that certain changes and modifications arecomprehended by the disclosure and may be practiced without undueexperimentation within the scope of the appended claims, which arepresented by way of illustration not limitation.

1 105 1 36 PRT Homo sapiens 1 Tyr Pro Ile Lys Pro Glu Ala Pro Gly GluAsp Ala Ser Pro Glu Glu 1 5 10 15 Leu Asn Arg Tyr Tyr Ala Ser Leu ArgHis Tyr Leu Asn Leu Val Thr 20 25 30 Arg Gln Arg Tyr 35 2 34 PRT Homosapiens 2 Ile Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu LeuAsn 1 5 10 15 Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val ThrArg Gln 20 25 30 Arg Tyr 3 15 PRT Homo sapiens 3 Ala Ser Leu Arg His TyrLeu Asn Leu Val Thr Arg Gln Arg Tyr 1 5 10 15 4 33 PRT Homo sapiens 4Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu Asn Arg 1 5 1015 Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg 20 2530 Tyr 5 32 PRT Homo sapiens 5 Pro Glu Ala Pro Gly Glu Asp Ala Ser ProGlu Glu Leu Asn Arg Tyr 1 5 10 15 Tyr Ala Ser Leu Arg His Tyr Leu AsnLeu Val Thr Arg Gln Arg Tyr 20 25 30 6 31 PRT Homo sapiens 6 Glu Ala ProGly Glu Asp Ala Ser Pro Glu Glu Leu Asn Arg Tyr Tyr 1 5 10 15 Ala SerLeu Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr 20 25 30 7 30 PRTHomo sapiens 7 Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu Asn Arg TyrTyr Ala 1 5 10 15 Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln ArgTyr 20 25 30 8 29 PRT Homo sapiens 8 Pro Gly Glu Asp Ala Ser Pro Glu GluLeu Asn Arg Tyr Tyr Ala Ser 1 5 10 15 Leu Arg His Tyr Leu Asn Leu ValThr Arg Gln Arg Tyr 20 25 9 28 PRT Homo sapiens 9 Gly Glu Asp Ala SerPro Glu Glu Leu Asn Arg Tyr Tyr Ala Ser Leu 1 5 10 15 Arg His Tyr LeuAsn Leu Val Thr Arg Gln Arg Tyr 20 25 10 27 PRT Homo sapiens 10 Glu AspAla Ser Pro Glu Glu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg 1 5 10 15 HisTyr Leu Asn Leu Val Thr Arg Gln Arg Tyr 20 25 11 26 PRT Homo sapiens 11Asp Ala Ser Pro Glu Glu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His 1 5 1015 Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr 20 25 12 25 PRT Homo sapiens12 Ala Ser Pro Glu Glu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr 1 510 15 Leu Asn Leu Val Thr Arg Gln Arg Tyr 20 25 13 24 PRT Homo sapiens13 Ser Pro Glu Glu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu 1 510 15 Asn Leu Val Thr Arg Gln Arg Tyr 20 14 23 PRT Homo sapiens 14 ProGlu Glu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn 1 5 10 15Leu Val Thr Arg Gln Arg Tyr 20 15 22 PRT Homo sapiens 15 Glu Glu Leu AsnArg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu 1 5 10 15 Val Thr ArgGln Arg Tyr 20 16 21 PRT Homo sapiens 16 Glu Leu Asn Arg Tyr Tyr Ala SerLeu Arg His Tyr Leu Asn Leu Val 1 5 10 15 Thr Arg Gln Arg Tyr 20 17 20PRT Homo sapiens 17 Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu AsnLeu Val Thr 1 5 10 15 Arg Gln Arg Tyr 20 18 19 PRT Homo sapiens 18 AsnArg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg 1 5 10 15Gln Arg Tyr 19 18 PRT Homo sapiens 19 Arg Tyr Tyr Ala Ser Leu Arg HisTyr Leu Asn Leu Val Thr Arg Gln 1 5 10 15 Arg Tyr 20 17 PRT Homo sapiens20 Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg 1 510 15 Tyr 21 16 PRT Homo sapiens 21 Tyr Ala Ser Leu Arg His Tyr Leu AsnLeu Val Thr Arg Gln Arg Tyr 1 5 10 15 22 36 PRT Homo sapiens 22 Tyr ProSer Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp 1 5 10 15 MetAla Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr 20 25 30 ArgGln Arg Tyr 35 23 11 PRT Homo sapiens 23 His Tyr Ile Asn Leu Ile Thr ArgGln Arg Tyr 1 5 10 24 34 PRT Homo sapiens 24 Ser Lys Pro Asp Asn Pro GlyGlu Asp Ala Pro Ala Glu Asp Met Ala 1 5 10 15 Arg Tyr Tyr Ser Ala LeuArg His Tyr Ile Asn Leu Ile Thr Arg Gln 20 25 30 Arg Tyr 25 33 PRT Homosapiens 25 Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp Met AlaArg 1 5 10 15 Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr ArgGln Arg 20 25 30 Tyr 26 32 PRT Homo sapiens 26 Pro Asp Asn Pro Gly GluAsp Ala Pro Ala Glu Asp Met Ala Arg Tyr 1 5 10 15 Tyr Ser Ala Leu ArgHis Tyr Ile Asn Leu Ile Thr Arg Gln Arg Tyr 20 25 30 27 31 PRT Homosapiens 27 Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp Met Ala Arg TyrTyr 1 5 10 15 Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln ArgTyr 20 25 30 28 30 PRT Homo sapiens 28 Asn Pro Gly Glu Asp Ala Pro AlaGlu Asp Met Ala Arg Tyr Tyr Ser 1 5 10 15 Ala Leu Arg His Tyr Ile AsnLeu Ile Thr Arg Gln Arg Tyr 20 25 30 29 29 PRT Homo sapiens 29 Pro GlyGlu Asp Ala Pro Ala Glu Asp Met Ala Arg Tyr Tyr Ser Ala 1 5 10 15 LeuArg His Tyr Ile Asn Leu Ile Thr Arg Gln Arg Tyr 20 25 30 28 PRT Homosapiens 30 Gly Glu Asp Ala Pro Ala Glu Asp Met Ala Arg Tyr Tyr Ser AlaLeu 1 5 10 15 Arg His Tyr Ile Asn Leu Ile Thr Arg Gln Arg Tyr 20 25 3127 PRT Homo sapiens 31 Glu Asp Ala Pro Ala Glu Asp Met Ala Arg Tyr TyrSer Ala Leu Arg 1 5 10 15 His Tyr Ile Asn Leu Ile Thr Arg Gln Arg Tyr 2025 32 26 PRT Homo sapiens 32 Asp Ala Pro Ala Glu Asp Met Ala Arg Tyr TyrSer Ala Leu Arg His 1 5 10 15 Tyr Ile Asn Leu Ile Thr Arg Gln Arg Tyr 2025 33 25 PRT Homo sapiens 33 Ala Pro Ala Glu Asp Met Ala Arg Tyr Tyr SerAla Leu Arg His Tyr 1 5 10 15 Ile Asn Leu Ile Thr Arg Gln Arg Tyr 20 2534 24 PRT Homo sapiens 34 Pro Ala Glu Asp Met Ala Arg Tyr Tyr Ser AlaLeu Arg His Tyr Ile 1 5 10 15 Asn Leu Ile Thr Arg Gln Arg Tyr 20 35 23PRT Homo sapiens 35 Ala Glu Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg HisTyr Ile Asn 1 5 10 15 Leu Ile Thr Arg Gln Arg Tyr 20 36 22 PRT Homosapiens 36 Glu Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile AsnLeu 1 5 10 15 Ile Thr Arg Gln Arg Tyr 20 37 21 PRT Homo sapiens 37 AspMet Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile 1 5 10 15Thr Arg Gln Arg Tyr 20 38 20 PRT Homo sapiens 38 Met Ala Arg Tyr Tyr SerAla Leu Arg His Tyr Ile Asn Leu Ile Thr 1 5 10 15 Arg Gln Arg Tyr 20 3919 PRT Homo sapiens 39 Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile AsnLeu Ile Thr Arg 1 5 10 15 Gln Arg Tyr 40 18 PRT Homo sapiens 40 Arg TyrTyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln 1 5 10 15 ArgTyr 41 17 PRT Homo sapiens 41 Tyr Tyr Ser Ala Leu Arg His Tyr Ile AsnLeu Ile Thr Arg Gln Arg 1 5 10 15 Tyr 42 16 PRT Homo sapiens 42 Tyr SerAla Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln Arg Tyr 1 5 10 15 43 15PRT Homo sapiens 43 Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg GlnArg Tyr 1 5 10 15 44 14 PRT Homo sapiens 44 Ala Leu Arg His Tyr Ile AsnLeu Ile Thr Arg Gln Arg Tyr 1 5 10 45 13 PRT Homo sapiens 45 Leu Arg HisTyr Ile Asn Leu Ile Thr Arg Gln Arg Tyr 1 5 10 46 12 PRT Homo sapiens 46Arg His Tyr Ile Asn Leu Ile Thr Arg Gln Arg Tyr 1 5 10 47 36 PRT Homosapiens 47 Ala Ser Leu Glu Pro Glu Tyr Pro Gly Asp Asn Ala Thr Pro GluGln 1 5 10 15 Met Ala Gln Tyr Ala Ala Glu Leu Arg Arg Tyr Ile Asn MetLeu Thr 20 25 30 Arg Pro Arg Tyr 35 48 11 PRT Homo sapiens 48 Arg TyrIle Asn Met Leu Thr Arg Pro Arg Tyr 1 5 10 49 34 PRT Homo sapiens 49 LeuGlu Pro Glu Tyr Pro Gly Asp Asn Ala Thr Pro Glu Gln Met Ala 1 5 10 15Gln Tyr Ala Ala Glu Leu Arg Arg Tyr Ile Asn Met Leu Thr Arg Pro 20 25 30Arg Tyr 50 33 PRT Homo sapiens 50 Glu Pro Glu Tyr Pro Gly Asp Asn AlaThr Pro Glu Gln Met Ala Gln 1 5 10 15 Tyr Ala Ala Glu Leu Arg Arg TyrIle Asn Met Leu Thr Arg Pro Arg 20 25 30 Tyr 51 32 PRT Homo sapiens 51Pro Glu Tyr Pro Gly Asp Asn Ala Thr Pro Glu Gln Met Ala Gln Tyr 1 5 1015 Ala Ala Glu Leu Arg Arg Tyr Ile Asn Met Leu Thr Arg Pro Arg Tyr 20 2530 52 31 PRT Homo sapiens 52 Glu Tyr Pro Gly Asp Asn Ala Thr Pro Glu GlnMet Ala Gln Tyr Ala 1 5 10 15 Ala Glu Leu Arg Arg Tyr Ile Asn Met LeuThr Arg Pro Arg Tyr 20 25 30 53 30 PRT Homo sapiens 53 Tyr Pro Gly AspAsn Ala Thr Pro Glu Gln Met Ala Gln Tyr Ala Ala 1 5 10 15 Glu Leu ArgArg Tyr Ile Asn Met Leu Thr Arg Pro Arg Tyr 20 25 30 54 29 PRT Homosapiens 54 Pro Gly Asp Asn Ala Thr Pro Glu Gln Met Ala Gln Tyr Ala AlaGlu 1 5 10 15 Leu Arg Arg Tyr Ile Asn Met Leu Thr Arg Pro Arg Tyr 20 2555 28 PRT Homo sapiens 55 Gly Asp Asn Ala Thr Pro Glu Gln Met Ala GlnTyr Ala Ala Glu Leu 1 5 10 15 Arg Arg Tyr Ile Asn Met Leu Thr Arg ProArg Tyr 20 25 56 27 PRT Homo sapiens 56 Asp Asn Ala Thr Pro Glu Gln MetAla Gln Tyr Ala Ala Glu Leu Arg 1 5 10 15 Arg Tyr Ile Asn Met Leu ThrArg Pro Arg Tyr 20 25 57 26 PRT Homo sapiens 57 Asn Ala Thr Pro Glu GlnMet Ala Gln Tyr Ala Ala Glu Leu Arg Arg 1 5 10 15 Tyr Ile Asn Met LeuThr Arg Pro Arg Tyr 20 25 58 25 PRT Homo sapiens 58 Ala Thr Pro Glu GlnMet Ala Gln Tyr Ala Ala Glu Leu Arg Arg Tyr 1 5 10 15 Ile Asn Met LeuThr Arg Pro Arg Tyr 20 25 59 24 PRT Homo sapiens 59 Thr Pro Glu Gln MetAla Gln Tyr Ala Ala Glu Leu Arg Arg Tyr Ile 1 5 10 15 Asn Met Leu ThrArg Pro Arg Tyr 20 60 23 PRT Homo sapiens 60 Pro Glu Gln Met Ala Gln TyrAla Ala Glu Leu Arg Arg Tyr Ile Asn 1 5 10 15 Met Leu Thr Arg Pro ArgTyr 20 61 22 PRT Homo sapiens 61 Glu Gln Met Ala Gln Tyr Ala Ala Glu LeuArg Arg Tyr Ile Asn Met 1 5 10 15 Leu Thr Arg Pro Arg Tyr 20 62 21 PRTHomo sapiens 62 Gln Met Ala Gln Tyr Ala Ala Glu Leu Arg Arg Tyr Ile AsnMet Leu 1 5 10 15 Thr Arg Pro Arg Tyr 20 63 20 PRT Homo sapiens 63 MetAla Gln Tyr Ala Ala Glu Leu Arg Arg Tyr Ile Asn Met Leu Thr 1 5 10 15Arg Pro Arg Tyr 20 64 19 PRT Homo sapiens 64 Ala Gln Tyr Ala Ala Glu LeuArg Arg Tyr Ile Asn Met Leu Thr Arg 1 5 10 15 Pro Arg Tyr 65 18 PRT Homosapiens 65 Gln Tyr Ala Ala Glu Leu Arg Arg Tyr Ile Asn Met Leu Thr ArgPro 1 5 10 15 Arg Tyr 66 17 PRT Homo sapiens 66 Tyr Ala Ala Glu Leu ArgArg Tyr Ile Asn Met Leu Thr Arg Pro Arg 1 5 10 15 Tyr 67 16 PRT Homosapiens 67 Ala Ala Glu Leu Arg Arg Tyr Ile Asn Met Leu Thr Arg Pro ArgTyr 1 5 10 15 68 15 PRT Homo sapiens 68 Ala Glu Leu Arg Arg Tyr Ile AsnMet Leu Thr Arg Pro Arg Tyr 1 5 10 15 69 14 PRT Homo sapiens 69 Glu LeuArg Arg Tyr Ile Asn Met Leu Thr Arg Pro Arg Tyr 1 5 10 70 13 PRT Homosapiens 70 Leu Arg Arg Tyr Ile Asn Met Leu Thr Arg Pro Arg Tyr 1 5 10 7112 PRT Homo sapiens 71 Arg Arg Tyr Ile Asn Met Leu Thr Arg Pro Arg Tyr 15 10 72 36 PRT Rat 72 Tyr Pro Ala Lys Pro Glu Ala Pro Gly Glu Asp AlaSer Pro Glu Glu 1 5 10 15 Leu Ser Arg Tyr Tyr Ala Ser Leu Arg His TyrLeu Asn Leu Val Thr 20 25 30 Arg Gln Arg Tyr 35 73 36 PRT Pig 73 Tyr ProAla Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu 1 5 10 15 LeuSer Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr 20 25 30 ArgGln Arg Tyr 35 74 36 PRT Guinea pig 74 Tyr Pro Ser Lys Pro Glu Ala ProGly Ser Asp Ala Ser Pro Glu Glu 1 5 10 15 Leu Ala Arg Tyr Tyr Ala SerLeu Arg His Tyr Leu Asn Leu Val Thr 20 25 30 Arg Gln Arg Tyr 35 75 36PRT Rat 75 Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala GluAsp 1 5 10 15 Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn LeuIle Thr 20 25 30 Arg Gln Arg Tyr 35 76 36 PRT Rabbit 76 Tyr Pro Ser LysPro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp 1 5 10 15 Met Ala ArgTyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr 20 25 30 Arg Gln ArgTyr 35 77 36 PRT Dog 77 Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp AlaPro Ala Glu Asp 1 5 10 15 Met Ala Arg Tyr Tyr Ser Ala Leu Arg His TyrIle Asn Leu Ile Thr 20 25 30 Arg Gln Arg Tyr 35 78 36 PRT Pig 78 Tyr ProSer Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp 1 5 10 15 LeuAla Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr 20 25 30 ArgGln Arg Tyr 35 79 36 PRT Cow 79 Tyr Pro Ser Lys Pro Asp Asn Pro Gly GluAsp Ala Pro Ala Glu Asp 1 5 10 15 Leu Ala Arg Tyr Tyr Ser Ala Leu ArgHis Tyr Ile Asn Leu Ile Thr 20 25 30 Arg Gln Arg Tyr 35 80 36 PRT Sheep80 Tyr Pro Ser Lys Pro Asp Asn Pro Gly Asp Asp Ala Pro Ala Glu Asp 1 510 15 Leu Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr 2025 30 Arg Gln Arg Tyr 35 81 36 PRT Guinea pig 81 Tyr Pro Ser Lys Pro AspAsn Pro Gly Glu Asp Ala Pro Ala Glu Asp 1 5 10 15 Met Ala Arg Tyr TyrSer Ala Leu Arg His Tyr Ile Asn Leu Ile Thr 20 25 30 Arg Gln Arg Tyr 3582 36 PRT Sheep 82 Ala Pro Leu Glu Pro Val Tyr Pro Gly Asp Asn Ala ThrPro Glu Gln 1 5 10 15 Met Ala Gln Tyr Ala Ala Asp Leu Arg Arg Tyr IleAsn Met Leu Thr 20 25 30 Arg Pro Arg Tyr 35 83 36 PRT Pig 83 Ala Pro LeuGlu Pro Val Tyr Pro Gly Asp Asp Ala Thr Pro Glu Gln 1 5 10 15 Met AlaGln Tyr Ala Ala Glu Leu Arg Arg Tyr Ile Asn Met Leu Thr 20 25 30 Arg ProArg Tyr 35 84 36 PRT Dog 84 Ala Pro Leu Glu Pro Val Tyr Pro Gly Asp AspAla Thr Pro Glu Gln 1 5 10 15 Met Ala Gln Tyr Ala Ala Glu Leu Arg ArgTyr Ile Asn Met Leu Thr 20 25 30 Arg Pro Arg Tyr 35 85 36 PRT Cat 85 AlaPro Leu Glu Pro Val Tyr Pro Gly Asp Asn Ala Thr Pro Glu Gln 1 5 10 15Met Ala Gln Tyr Ala Ala Glu Leu Arg Arg Tyr Ile Asn Met Leu Thr 20 25 30Arg Pro Arg Tyr 35 86 36 PRT Cow 86 Ala Pro Leu Glu Pro Glu Tyr Pro GlyAsp Asp Ala Thr Pro Glu Gln 1 5 10 15 Met Ala Gln Tyr Ala Ala Glu LeuArg Arg Tyr Ile Asn Met Leu Thr 20 25 30 Arg Pro Arg Tyr 35 87 36 PRTRat 87 Ala Pro Leu Glu Pro Met Tyr Pro Gly Asp Tyr Ala Thr His Glu Gln 15 10 15 Arg Ala Gln Tyr Glu Thr Gln Leu Arg Arg Tyr Ile Asn Thr Leu Thr20 25 30 Arg Pro Arg Tyr 35 88 36 PRT mouse 88 Ala Pro Leu Glu Pro MetTyr Pro Gly Asp Tyr Ala Thr Pro Glu Gln 1 5 10 15 Met Ala Gln Tyr GluThr Gln Leu Arg Arg Tyr Ile Asn Thr Leu Thr 20 25 30 Arg Pro Arg Tyr 3589 37 PRT Guinea pig 89 Ala Pro Leu Glu Pro Val Tyr Pro Gly Asp Asn AlaThr Pro Glu Gln 1 5 10 15 Gln Met Ala Gln Tyr Ala Ala Glu Met Arg ArgTyr Ile Asn Met Leu 20 25 30 Thr Arg Pro Arg Tyr 35 90 22 PRT Homosapiens 90 Asp Glu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu AsnLeu 1 5 10 15 Val Thr Arg Gln Arg Tyr 20 91 24 PRT Homo sapiens 91 ThrPro Glu Glu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu 1 5 10 15Asn Leu Val Thr Arg Gln Arg Tyr 20 92 25 PRT Homo sapiens 92 Val Ser ProGlu Glu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr 1 5 10 15 Leu AsnLeu Val Thr Arg Gln Arg Tyr 20 25 93 26 PRT Homo sapiens 93 Glu Ala SerPro Glu Glu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His 1 5 10 15 Tyr LeuAsn Leu Val Thr Arg Gln Arg Tyr 20 25 94 27 PRT Homo sapiens 94 Asp AspAla Ser Pro Glu Glu Leu Asn Arg Tyr Tyr Ala Ser Leu Arg 1 5 10 15 HisTyr Leu Asn Leu Val Thr Arg Gln Arg Tyr 20 25 95 30 PRT Homo sapiens 95Val Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu Asn Arg Tyr Tyr Ala 1 5 1015 Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr 20 25 30 9631 PRT Homo sapiens 96 Asp Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu LeuAsn Arg Tyr Tyr 1 5 10 15 Ala Ser Leu Arg His Tyr Leu Asn Leu Val ThrArg Gln Arg Tyr 20 25 30 97 33 PRT Homo sapiens 97 Gln Pro Glu Ala ProGly Glu Asp Ala Ser Pro Glu Glu Leu Asn Arg 1 5 10 15 Tyr Tyr Ala SerLeu Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg 20 25 30 Tyr 98 33 PRTHomo sapiens 98 Arg Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu LeuAsn Arg 1 5 10 15 Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val ThrArg Gln Arg 20 25 30 Tyr 99 33 PRT Homo sapiens 99 Asn Pro Glu Ala ProGly Glu Asp Ala Ser Pro Glu Glu Leu Asn Arg 1 5 10 15 Tyr Tyr Ala SerLeu Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg 20 25 30 Tyr 100 34 PRTHomo sapiens 100 Val Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu GluLeu Asn 1 5 10 15 Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu ValThr Arg Gln 20 25 30 Arg Tyr 101 34 PRT Homo sapiens 101 Leu Lys Pro GluAla Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu Asn 1 5 10 15 Arg Tyr TyrAla Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln 20 25 30 Arg Tyr 10227 PRT Homo sapiens 102 Asp Asp Ala Ser Pro Asp Glu Leu Asn Arg Tyr TyrAla Ser Leu Arg 1 5 10 15 His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr 2025 103 31 PRT Homo sapiens 103 Asp Ala Pro Gly Glu Asp Ala Thr Pro GluGlu Leu Asn Arg Tyr Tyr 1 5 10 15 Ala Ser Leu Arg His Tyr Leu Asn LeuVal Thr Arg Gln Arg Tyr 20 25 30 104 33 PRT Homo sapiens 104 Asn Pro GluAla Pro Gly Glu Asp Ala Ser Pro Asp Glu Leu Asn Arg 1 5 10 15 Tyr TyrAla Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg 20 25 30 Tyr 10534 PRT Homo sapeins 105 Leu Lys Pro Glu Ala Pro Gly Asp Asp Ala Ser ProGlu Glu Leu Asn 1 5 10 15 Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu AsnLeu Val Thr Arg Gln 20 25 30 Arg Tyr

What is claimed is:
 1. A peptide YY (PYY) composition comprised of asolubilizing agent, a chelating agent, a polyol and a PYY peptidewherein the PYY peptide is comprised of an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 2, 3 and SEQ ID NOs: 90-105. 2.The PYY composition of claim 1 wherein the PYY composition is comprisedof at least two polyols.
 3. The PYY composition of claim 2 wherein thepolyols are selected from the group consisting of sucrose, mannitol,sorbitol, lactose, L-arabinose, D-erythrose, D-ribose, D-xylose,D-mannose, trehalose, D-galactose, lactulose, cellobiose, gentibiose,glycerin and polyethylene glycol.
 4. The PYY composition of claim 3wherein the polyols are lactose and sorbitol.
 5. The PYY compostion ofclaim 1 wherein the chelating agent is ethylene diamine tetraacetic acid(EDTA) or ethylene glycol tetraacetic acid (EGTA).
 6. The PYYcomposition of claim 5 wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA).
 7. The PYY composition of claim 1 wherein thesolubilizing agent is selected from the group consisting of acyclodextran, hydroxypropyl-p-cyclodextran,sulfobutylether-β-cyclodextran and methyl-p-cyclodextrin.
 8. The PYYcomposition of claim 7 wherein the solubilizing agent is a cyclodextrin.9. The PYY composition of claims 1 wherein the composition is furthercomprised of a surface-active agent.
 10. The PYY composition of claim 9wherein the surface-active agent is selected from the group consistingof nonionic polyoxyethylene ether, bile salts such, sodium glycocholate(SGC), deoxycholate (DOC), derivatives of fusidic acid, sodiumtaurodihydrofusidate (STDHF), L-α-phospharidycholine didecanoyl (DDPC)polysorbate 80 and polysorbate 20,), polyethylene glycol (PEG), cetylalcohol, polyvinylpyrolidone (PVP), polyvinyl alcohol (PVA), lanolinalcohol, and sorbitan monooleate.
 11. The PYY composition of claim 10wherein the surface-active agent is L-α-Phospharidycholine didecanoyl(DDPC).
 12. The PYY composition of claim 1 wherein the composition is anaqueous PYY composition further comprised of water.
 13. The PYYcomposition of claim 12 wherein the aqueous PYY composition has a pH ofabout of about 3-6.
 14. The PYY composition of claim 13 wherein the pHof the aqueous PYY composition is 5.0±0.3.
 15. The PYY composition ofclaim 1 wherein the PYY peptide is comprised of PYY(3-36) (SEQ ID NO:2).
 16. A PYY composition comprised of a solubilizing agent, a chelatingagent, a polyol, a surface active agent and a PYY peptide wherein thePYY peptide is comprised of an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 2, 3 and SEQ ID NOs: 90-105.
 17. The PYYcomposition of claim 16 wherein the composition is further comprised ofat least two polyols.
 18. The PYY composition of claim 17 wherein thepolyols are selected from the group consisting of sucrose, mannitol,sorbitol, lactose, L-arabinose, D-erythrose, D-ribose, D-xylose,D-mannose, trehalose, D-galactose, lactulose, cellobiose, gentibiose,glycerin and polyethylene glycol.
 19. The PYY composition of claim 18wherein the polyols are lactose and sorbitol.
 20. The PYY composition ofclaim 16 wherein the chelating agent is ethylene diamine tetraaceticacid (EDTA) or ethylene glycol tetraacetic acid (EGTA).
 21. The PYYcomposition of claim 20 wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA).
 22. The PYY composition of claim 16 wherein thesolubilizing agent is selected from the group consisting of acyclodextran, hydroxypropyl-β-cyclodextran,sulfobutylether-β-cyclodextran and methyl-β-cyclodextrin.
 23. The PYYcomposition of claim 22 wherein the solubilizing agent is amethyl-β-cyclodextrin.
 24. The PYY composition of claim 16 wherein thesurface-active agent is selected from the group consisting of nonionicpolyoxyethylene ether, bile salts such, sodium glycocholate (SGC),deoxycholate (DOC), derivatives of fusidic acid, sodiumtaurodihydrofusidate (STDHF), L-α-phospharidycholine didecanoyl (DDPC)polysorbate 80 and polysorbate 20,), polyethylene glycol (PEG), cetylalcohol, polyvinylpyrolidone (PVP), polyvinyl alcohol (PVA), lanolinalcohol, and sorbitan monooleate.
 25. The PYY composition of claim 24wherein the surface-active agent is L-α-phospharidycholine didecanoyl(DDPC).
 26. The PYY composition of claim 16 wherein the composition isan aqueous PYY composition further comprised of water.
 27. The PYYcomposition of claim 26 wherein the aqueous PYY composition has a pH ofabout of about 3-6.
 28. The PYY composition of claim 27 wherein the pHof the aqueous PYY composition is 5.0+0.3.
 29. The PYY composition ofclaim 16 wherein the PYY peptide is comprised of PYY(3-36) (SEQ ID NO:2).
 30. A PYY composition comprised of methyl-β-cyclodextrin, EDTA,lactose, sorbitol, DDPC and a PYY peptide wherein the PYY peptide iscomprised of an amino acid sequence selected from the group consistingof SEQ ID NOs: 2, 3 and SEQ ID NOs: 90-105.
 31. The PYY composition ofclaim 30 wherein the PYY peptide is comprised of PYY(3-36) (SEQ ID NO:2).
 32. A PYY composition comprised of water, sodium citrate, citricacid, methyl-β-cyclodextrin, EDTA, lactose, sorbitol, DDPC and PYY(3-36)(SEQ ID NO: 2), wherin the formulation has a pH of about 5.0+0.3. 33.The PYY composition of claim 32 further comprised of a preservativeselected from the group consisting of chlorobutanol and benzalkoniumchloride.
 34. The composition of claim 33 wherein the preservative ischlorobutanol.
 35. A PYY composition comprised of water, chlorobutanol,sodium citrate, citric acid, methyl-β-cyclodextrin, EDTA, lactose,sorbitol, DDPC and PYY(3-36) (SEQ ID NO: 2), wherein the formulation hasa pH of about 5.0±0.3.
 36. A PYY composition comprised of water,benzalkonium chloride, sodium citrate, citric acid,methyl-β-cyclodextrin, EDTA, lactose, sorbitol, DDPC and PYY(3-36) (SEQID NO: 2), wherein the formulation has a pH of about 5.0±0.3.
 37. A PYYcomposition comprised of water, chlorobutanol at a concentration of 5mg/ml, sodium citrate at a concentration of 1.62 mg/ml, citric acid at aconcentration of 0.86 mg/ml, methyl-β-cyclodextrin at a concentration of45 mg/ml, EDTA at a concentration of 1 mg/ml, lactose at a concentrationof 9 mg/ml, sorbitol at a concentration of 18.2 mg/ml, DDPC at aconcentration of 1 mg/ml and PYY(3-36) (SEQ ID NO: 2) at a concentrationof 1 mg/ml, wherein the formulation has a pH of about 5.0±0.3.
 38. A PYYcomposition comprised of water, chlorobutanol at a concentration of 5mg/ml, sodium citrate at a concentration of 1.62 mg/ml, citric acid at aconcentration of 0.86 mg/ml, methyl-β-cyclodextrin at a concentration of45 mg/ml, EDTA at a concentration of 1 mg/ml, lactose at a concentrationof 9 mg/ml, sorbitol at a concentration of 18.2 mg/ml, DDPC at aconcentration of 1 mg/ml and PYY(3-36) (SEQ ID NO: 2) at a concentrationof 15 mg/ml, wherein the formulation has a pH of about 5.0±0.3.